U.S. patent number 7,807,815 [Application Number 11/174,453] was granted by the patent office on 2010-10-05 for compositions comprising immunostimulatory sirna molecules and dlindma or dlendma.
This patent grant is currently assigned to Protiva Biotherapeutics, Inc.. Invention is credited to James Heyes, Adam Judge, Ian MacLachlan, Lorne Palmer.
United States Patent |
7,807,815 |
MacLachlan , et al. |
October 5, 2010 |
Compositions comprising immunostimulatory siRNA molecules and
DLinDMA or DLenDMA
Abstract
The present invention provides siRNA molecules and methods of
using such siRNA molecules to modulate an immune response and to
silence expression of a target gene.
Inventors: |
MacLachlan; Ian (Mission,
CA), Judge; Adam (Vancouver, CA), Heyes;
James (Burnaby, CA), Palmer; Lorne (Vancouver,
CA) |
Assignee: |
Protiva Biotherapeutics, Inc.
(Burnaby, BC, CA)
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Family
ID: |
35786796 |
Appl.
No.: |
11/174,453 |
Filed: |
June 30, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060025366 A1 |
Feb 2, 2006 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60665297 |
Mar 25, 2005 |
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60627326 |
Nov 12, 2004 |
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60589363 |
Jul 19, 2004 |
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60585301 |
Jul 2, 2004 |
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Current U.S.
Class: |
536/24.5 |
Current CPC
Class: |
A61P
1/16 (20180101); A61P 37/02 (20180101); A61P
9/00 (20180101); A61P 37/06 (20180101); A61P
3/00 (20180101); C12N 15/111 (20130101); C12N
15/117 (20130101); A61P 43/00 (20180101); A61P
31/12 (20180101); A61P 35/00 (20180101); A61P
25/00 (20180101); A61P 29/00 (20180101); A61K
2039/55561 (20130101); C12N 2310/14 (20130101); C12N
2310/17 (20130101); C12N 2320/50 (20130101); A01K
2267/0331 (20130101) |
Current International
Class: |
C07H
21/04 (20060101) |
Field of
Search: |
;536/24.5 |
References Cited
[Referenced By]
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WO 2004/029212 |
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WO 2004/065546 |
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Aug 2004 |
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Jan 2005 |
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WO |
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WO 2005/019453 |
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Mar 2005 |
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WO |
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WO 2005/021044 |
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Mar 2005 |
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WO |
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WO 2005/026372 |
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Mar 2005 |
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WO 2007/048046 |
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WO |
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Primary Examiner: Angell; J. E
Attorney, Agent or Firm: Townsend and Townsend and Crew,
LLP
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application
Nos. 60/665,297, filed Mar. 25, 2005; 60/627,326, filed Nov. 12,
2004; 60/589,363, filed Jul. 19, 2004; and 60/585,301, filed Jul.
2, 2004, the disclosures of which are hereby incorporated by
reference in their entirety for all purposes.
Claims
What is claimed is:
1. A nucleic acid-lipid particle comprising: (a) a modified siRNA
comprising a double-stranded sequence of about 15 to about 30
nucleotides in length, said sequence comprising a
non-immunostimulatory mismatch motif relative to an unmodified
siRNA sequence that is capable of silencing expression of a target
sequence, wherein the mismatch motif consists of a 5'-XX'-3'
dinucleotide corresponding to a 5'-GU-3' dinucleotide in the sense
or antisense strand of the unmodified siRNA sequence, wherein X and
X' are independently selected from the group consisting of A, U, C,
and G, with the proviso that if X is G, X' is not U and if X' is U,
X is not G, wherein the modified siRNA is less immunogenic than the
unmodified siRNA sequence, and wherein the modified siRNA is
capable of silencing expression of the target sequence; (b) a
cationic lipid, wherein said cationic lipid is selected from the
group consisting of 1,2-DiLinoleyloxy-N,N-dimethylaminopropane
(DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
and a mixture thereof; (c) a non-cationic lipid; and (d) a
conjugated lipid that inhibits aggregation of particles.
2. The nucleic acid-lipid particle in accordance with claim 1,
wherein the modified siRNA has reduced toxicity relative to an
siRNA that is not in a nucleic acid-lipid particle.
3. The nucleic acid-lipid particle in accordance with claim 1,
wherein said cationic lipid is DLinDMA.
4. The nucleic acid-lipid particle in accordance with claim 1,
wherein said non-cationic lipid is a member selected from the group
consisting of distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-
phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol,
and a mixture thereof.
5. The nucleic acid-lipid particle in accordance with claim 1,
wherein said non-cationic lipid is DSPC.
6. The nucleic acid-lipid particle in accordance with claim 1,
wherein the conjugated lipid that inhibits aggregation of particles
comprises a polyethyleneglycol (PEG)-lipid and the PEG-lipid is
member selected from the group consisting of a PEG-diacylglycerol,
a PEG dialkyloxypropyl, a PEG-phospholipid, a PEG-ceramide, and a
mixture thereof.
7. The nucleic acid-lipid particle in accordance with claim 1,
wherein the conjugated lipid that inhibits aggregation of particles
comprises a PEG-dialkyloxypropyl (DAA) conjugate.
8. The nucleic acid-lipid particle in accordance with claim 7,
wherein the PEG-DAA conjugate is a member selected from the group
consisting of a PEG-dilauryloxypropyl (C.sub.12), a
PEG-dimyristyloxypropyl (C.sub.14), a PEG-dipalmityloxypropyl
(C.sub.16), and a PEG-distearyloxypropyl (C.sub.18).
9. The nucleic acid-lipid particle in accordance with claim 7,
wherein the PEG-DAA conjugate is a PEG-dimyristyloxypropyl
(C.sub.14).
10. The nucleic acid-lipid particle in accordance with claim 1,
wherein said cationic lipid comprises from about 15% to about 35%
of the total lipid present in said particle.
11. The nucleic acid-lipid particle in accordance with claim 1,
wherein said non-cationic lipid comprises from about 15% to about
25% of the total lipid present in said particle.
12. The nucleic acid-lipid particle in accordance with claim 7,
wherein said PEG-DAA conjugate comprises from 1% to about 10% of
the total lipid present in said particle.
13. The nucleic acid-lipid particle in accordance with claim 7,
wherein said PEG-DAA conjugate comprises about 2% of the total
lipid present in said particle.
14. The nucleic acid-lipid particle in accordance with claim 1,
further comprising cholesterol.
15. The nucleic acid-lipid particle in accordance with claim 14,
wherein the cholesterol comprises from about 40% to about 60% of
the total lipid present in said particle.
16. The nucleic acid-lipid particle in accordance with claim 1,
wherein the modified siRNA is fully encapsulated in said nucleic
acid-lipid particle.
17. A composition comprising a nucleic acid-lipid particle in
accordance with claim 1 and a carrier.
18. The nucleic acid-lipid particle in accordance with claim 1,
wherein said cationic lipid comprises from about 2% to about 60% of
the total lipid present in said particle.
19. The nucleic acid-lipid particle in accordance with claim 1,
wherein said cationic lipid comprises from about 40% to about 50%
of the total lipid present in said particle.
20. The nucleic acid-lipid particle in accordance with claim 1,
wherein said non-cationic lipid comprises from about 5% to about
90% of the total lipid present in said particle.
21. The nucleic acid-lipid particle in accordance with claim 1,
wherein said non-cationic lipid comprises from about 20% to about
85% of the total lipid present in said particle.
22. The nucleic acid-lipid particle in accordance with claim 14,
wherein the cholesterol comprises from about 20% to about 45% of
the total lipid present in said particle.
23. The nucleic acid-lipid particle in accordance with claim 1,
wherein said particle has a median diameter of from about 50 nm to
about 150 nm.
24. The nucleic acid-lipid particle in accordance with claim 1,
wherein said non-cationic lipid comprises a phospholipid and
cholesterol, and wherein said conjugated lipid that inhibits
aggregation of particles comprises a PEG-DAA conjugate.
25. The nucleic acid-lipid particle in accordance with claim 1,
wherein the modified siRNA further comprises a second
non-immunostimulatory mismatch motif.
26. The nucleic acid-lipid particle in accordance with claim 1,
wherein the modified siRNA comprises a double-stranded sequence of
about 19 to about 25 nucleotides in length.
27. The nucleic acid-lipid particle in accordance with claim 1,
wherein the modified siRNA comprises 3' overhangs.
28. The nucleic acid-lipid particle in accordance with claim 1,
wherein said conjugated lipid that inhibits aggregation of
particles comprises from about 1% to about 15% of the total lipid
present in said particle.
Description
BACKGROUND OF THE INVENTION
RNA interference (RNAi) is an evolutionarily conserved, sequence
specific mechanism triggered by double stranded RNA (dsRNA) that
induces degradation of complementary target single stranded mRNA
and "silencing" of the corresponding translated sequences (McManus
and Sharp, Nature Rev. Genet. 3:737 (2002)). RNAi functions by
enzymatic cleavage of longer dsRNA strands into biologically active
"short-interfering RNA" (siRNA) sequences of about 21-23
nucleotides in length (Elbashir, et al., Genes Dev. 15:188 (2001)).
siRNA can be used to downregulate or silence the transcription and
translation of a gene product of interest, i.e., a target
sequence.
Nucleic acids, like other macromolecules, can act as biological
response modifiers, i.e., can induce immune responses in mammals
upon in vivo administration. For example, poly(I:C)-LC has been
identified as a potent inducer of interferon (IFN) as well as a
macrophage activator and inducer of natural killer (NK) activity
(Talmadge et al., Cancer Res. 45:1058 (1985); Wiltrout et al., J.
Biol. Resp. Mod. 4:512 (1985); Krown, Sem. Oncol. 13:207 (1986);
and Ewel et al., Canc. Res. 52:3005 (1992)). Unfortunately, toxic
side effects have thus far prevented poly(I:C)-LC and other nucleic
acids from becoming a useful therapeutic agent.
Several phosphorothioate modified oligodeoxynucleotides (ODN) have
been reported to induce in vitro and in vivo B cell stimulation
(Tanaka et al., J. Exp. Med. 175:597 (1992); Branda et al.,
Biochem. Pharmacol. 45:2037 (1993); McIntyre et al., Antisense Res.
Develop. 3:309 (1993); and Pisetsky and Reich, Life Sciences 54:101
(1993)). However, none of these reports suggest a common structural
motif or sequence element in these ODN that might explain their
effects.
Recent reports have indicated that phosphorothioate-protected
single-stranded RNA sequences comprising a GU-rich sequence derived
from the U5 region of HIV-1 RNA complexed to the cationic lipid
1,2-dioleoyl-3-(trimethyammonium) (DOTAP) can induce expression of
the cytokines IL-6, IL-12p40, TNF-.alpha. and IFN-.alpha. (see,
e.g., Heil et al., Science 303:1526-1529 (2004)). In addition, U.S.
Patent Publication No. 20030232074 and WO 03/086280 describe
immunostimulatory RNA molecules, i.e., rRNA, tRNA, mRNA, and vRNA,
comprising at least one guanine and at least one uracil. However,
these reports do mention or suggest that siRNA has any
immunostimulatory properties or that siRNA can be used to modulate
an immune response.
Thus, there is a need for nucleic acid compositions that can be
modified to modulate (i.e., increase or decrease) their
immunostimulatory properties. The present invention addresses these
and other needs.
SUMMARY OF THE INVENTION
The present invention provides siRNA molecules and methods of using
such siRNA molecules to silence target gene expression and/or to
modulate (i.e., enhance or decrease) an immune response associated
with the siRNA molecules.
One embodiment of the invention provides a modified siRNA that is
capable of silencing expression of a target sequence, comprising a
double stranded region of about 15 to about 30 nucleotides in
length and a non-immunostimulatory mismatch motif consisting of a
5'-XX'-3' dinucleotide corresponding to a 5'-GU-3' dinucleotide in
an unmodified siRNA sequence that is capable of silencing
expression of the target sequence, wherein X and X' are
independently selected from the group consisting of A, U, C, and G,
with the proviso that if X is G, X' is not U and if X' is U, X is
not GU. The modified siRNA is less immunogenic than an siRNA that
does not comprise the non-immunostimulatory mismatch motif. In some
embodiments, the siRNA comprises one, two, three, or more
additional immunostimulatory mismatch motifs relative to the target
sequence. The immunostimulatory mismatch motifs may be adjacent to
each other or, alternatively, they may be separated by 1, 2, 4, 6,
8, 10, or 12 or more nucleotides.
Another embodiment of the invention provides a modified siRNA that
is capable of silencing expression of a target sequence comprising
a double stranded sequence of about 15 to about 30 nucleotides in
length and an immunostimulatory mismatch motif consisting of a
5'-GU-3' dinucleotide corresponding to a 5'-XX'-3' dinucleotide
motif in an unmodified siRNA that is capable of silencing
expression of a target sequence, wherein X and X' are independently
selected from the group consisting of A, U, C, and G, with the
proviso that if X is G, X' is not U and if X' is U, X is not GU.
The modified siRNA is more immunogenic than an siRNA that does not
comprise the immunostimulatory mismatch motif. In some embodiments,
the siRNA comprises one, two, three, or more additional
immunostimulatory mismatch motifs relative to the target sequence.
The immunostimulatory mismatch motifs may be adjacent to each other
or, alternatively, they may be separated by 1, 2, 4, 6, 8, 10, or
12 or more nucleotides.
In some embodiments, the siRNA described herein are used in methods
of silencing expression of a target sequence and/or in methods of
modulating (i.e., enhancing or reducing) immune responses
associated with the siRNA. An effective amount of the siRNA is
administered to a mammalian subject, thereby silencing expression
of a target sequence or modulating an immune response associated
with the siRNA.
The invention also provides pharmaceutical compositions comprising
the siRNA molecules described herein.
Another embodiment of the invention provides nucleic acid-lipid
particles comprising: the siRNA molecules described herein; a
cationic lipid; a non-cationic lipid; and a conjugated lipid that
inhibits aggregation of particles. The cationic lipid may be, e.g.,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), and N,N-dimethyl-2,3-dioleyloxy)propylamine
(DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), and
1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLendMA), or mixtures
thereof. The cationic lipid may comprise from about 2% to about
60%, about 5% to about 45%, about 5% to about 15%, or about 40% to
about 50% of the total lipid present in the particle.
The non-cationic lipid may be an anionic lipid or a neutral lipid
including, but not limited to, distearoylphosphatidylcholine
(DSPC), dioleoylphosphatidylcholine (DOPC),
dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol
(DOPG), dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or
mixtures thereof. The non-cationic lipid comprises from about 5% to
about 90% or about 20% to about 85% of the total lipid present in
the particle.
The conjugated lipid that inhibits aggregation of particles may be
a polyethyleneglycol (PEG)-lipid conjugate, a polyamide
(ATTA)-lipid conjugate, a cationic-polymer-lipid conjugates (CPLs),
or mixtures thereof. In one preferred embodiment, the nucleic
acid-lipid particules comprise either a PEG-lipid conjugate or an
ATTA-lipid conjugate together with a CPL. The conjugated lipid that
inhibits aggregation of particles may comprise a
polyethyleneglycol-lipid including, e.g., a PEG-diacylglycerol
(DAG), a PEG dialkyloxypropyl (DAA), a PEG-phospholipid, a
PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may
be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a
PEG-dipalmityloxypropyl (C16), and a PEG-distearyloxypropyl (C18).
In some embodiments, the conjugated lipid that inhibits aggregation
of particles has the formula: A-W-Y, wherein: A is a lipid moiety;
W is a hydrophilic polymer; and Y is a polycationic moiety. W may
be a polymer selected from the group consisting of
polyethyleneglycol (PEG), polyamide, polylactic acid, polyglycolic
acid, polylactic acid/polyglycolic acid copolymers or combinations
thereof, said polymer having a molecular weight of about 250 to
about 7000 daltons. In some embodiments, Y has at least 4 positive
charges at a selected pH. In some embodiments, Y may be lysine,
arginine, asparagine, glutamine, derivatives thereof and
combinations thereof. The conjugated lipid that prevents
aggregation of particles may be about 0% to about 20%, about 1% to
about 15%, about 4% to about 10%, or about 2% of the total lipid
present in said particle.
In some embodiments, the nucleic acid-lipid particle further
comprises cholesterol at, e.g., about 10% to about 60% or about 20%
to about 45% of the total lipid present in said particle.
In some embodiments, the siRNA in the nucleic acid-lipid particle
is not substantially degraded after exposure of the particle to a
nuclease at 37.degree. C. for at least 20, 30, 45, or 60 minutes;
or after incubation of the particle in serum at 37.degree. C. for
at least 30, 45, or 60 minutes.
In some embodiments, the siRNA is fully encapsulated in the nucleic
acid-lipid particle. In some embodiments, the siRNA is complexed to
the lipid portion of the particle.
The present invention further provides pharmaceutical compositions
comprising the nucleic acid-lipid particles described herein and a
pharmaceutically acceptable carrier.
Yet another embodiment of the invention provides a method of
modifying a siRNA having immunostimulatory properties. The method
comprises (a) providing an unmodified siRNA sequence comprising at
least one GU-rich motif (e.g., a 5'-GU-3' motif) and capable of
silencing expression of a target sequence; and (b) modifying the
siRNA to substitute the at least one GU-rich motif, with a
non-immunostimulatory mismatch motif relative to the target
sequence, wherein the non-immunostimulatory mismatch motif consists
of a 5'-XX'-3' motif corresponding to the at least one GU-rich
motif in the unmodified siRNA sequence, wherein X and X' are
independently selected from the group consisting of A, U, C, and G,
with the proviso that if X is G, X' is not U and if X' is U, X is
not G; thereby generating a modified siRNA that is less immunogenic
than the unmodified siRNA sequence and is capable of silencing
expression of the target sequence.
Another embodiment of the invention provides a method of modifying
an siRNA having non-immunostimulatory properties. The method
comprises (a) providing an unmodified siRNA lacking a GU-rich motif
(e.g., a 5'-GU-3' motif) and capable of silencing expression of a
target sequence; and (b) modifying the siRNA to introduce at least
one immunostimulatory mismatch motif relative to the target
sequence, wherein the at least one immunostimulatory mismatch motif
consists of a GU-rich motif (e.g., a 5'-GU-3' dinucleotide motif)
corresponding to a 5'-XX'-3' motif in the unmodified siRNA, wherein
X and X' are independently selected from the group consisting of A,
U, C, and G, with the proviso that if X is G, X' is not U and if X'
is U, X is not G; thereby generating a modified siRNA that is more
immunogenic than the unmodified siRNA and is capable of silencing
expression of the target sequence.
A further embodiment of the invention provides a method of
identifying and/or modifying an siRNA having immunostimulatory
properties. The method comprises (a) providing a target nucleic
acid sequence; (b) identifying a siRNA sequence that is
complementary to the target sequence and comprises at least one
GU-rich motif (e.g., a 5'-GU-3' motif), wherein the presence of the
at least one GU-rich motif identifies an immunostimulatory siRNA;
and (c) contacting the modified siRNA sequence with a mammalian
responder cell under conditions suitable for said responder cell to
produce a detectable immune response. In some embodiments, the
siRNA is modified substituting the at least one GU-rich motif with
a non-immunostimulatory mismatch motif relative to the target
sequence, wherein the non-immunostimulatory mismatch motif consists
of a 5'-XX'-3' motif corresponding to the at least one GU-rich
motif in the unmodified siRNA sequence, wherein X and X' are
independently selected from the group consisting of A, U, C, and G,
with the proviso that if X is G, X' is not U and if X' is U, X is
not G, thereby generating a modified siRNA that is less immunogenic
than the unmodified siRNA sequence. In some embodiments, the method
further comprises contacting the unmodified siRNA sequence with a
mammalian responder cell (e.g., a peripheral blood mononuclear
cell) under conditions suitable for the responder cell to produce a
detectable immune response; and comparing the immune response(e.g.,
production of a cytokine or growth factor such as, e.g.,
TNF-.alpha., IFN-.alpha., IL-6, IL-12, and combinations thereof)
produced by the modified siRNA with the immune response produced by
the unmodified siRNA. The mammalian responder cell may be from a
naive mammal (i.e., a mammal that has not previously been in
contact with the gene product of the target nucleic acid
sequence).
Even another embodiment of the invention provides a method of
identifying and/or modifying an siRNA having non-immunostimulatory
properties. The method comprises (a) providing a target nucleic
acid sequence; (b) identifying a siRNA sequence that is
complementary to the target sequence and lacks a 5'-GU-3' motif,
wherein the absence of the 5'-GU-3' motif identifies a
non-immunostimulatory siRNA; and (c) contacting the modified siRNA
sequence with a mammalian responder cell under conditions suitable
for the responder cell to produce a detectable immune response. In
some embodiments, the method further comprises modifying the siRNA
to introduce at least one immunostimulatory mismatch motif relative
to the target sequence, wherein the at least one immunostimulatory
mismatch motif consists of a 5'-GU-3' dinucleotide corresponding to
a 5'-XX'-3' dinucleotide motif in the unmodified siRNA, wherein X
and X' are independently selected from the group consisting of A,
U, C, and G, with the proviso that if X is G, X' is not U and if X'
is U, X is not G, thereby generating a modified siRNA that is more
immunogenic than the unmodified siRNA. In some embodiments, the
method further comprises contacting the unmodified siRNA sequence
with a mammalian responder cell under conditions suitable for the
responder cell to produce a detectable immune response; and
comparing the immune response(e.g., production of a cytokine or
growth factor such as, e.g., TNF-.alpha., IFN-.alpha., IL-6, IL-12,
and combinations thereof) produced by the modified siRNA with the
immune response produced by the unmodified siRNA.
Even another embodiment of the invention provides isolated nucleic
acid molecules comprising a sequence set forth in Table 1, 2, 3, or
4.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates data demonstrating that SNALP encapsulating
siRNA exhibit extended blood circulation times.
FIG. 2 illustrates data demonstrating that SNALP encapsulating
siRNA can be programmed to target specific disease sites. FIG. 2A
illustrates data demonstrating targeting of SNALP to liver and FIG.
2B illustrates data demonstrating targeting of SNALP to tumors.
FIG. 3 illustrates data demonstrating that siRNA duplexes stimulate
production of type I interferons and inflammatory cytokines in
vitro and in vivo. FIG. 3A illustrates data demonstrating that
lipid-encapsulated siRNA induces IFN-.alpha. production in human
PBMC and lipid-complexed siRNA induces IL-6 and TNF-.alpha.
production in human PBMC. FIG. 3B illustrates data demonstrating
that lipid-encapsulated siRNA induces IFN-.alpha. production in
mice. FIG. 3C illustrates data demonstrating that
lipid-encapsulated siRNA induces IL-6 and TNF-.alpha. production in
mice. FIG. 3D illustrates data demonstrating that IFN-.alpha.
production in mice is dose dependent. FIG. 3E illustrates data
demonstrating that IFN-.alpha. production in human PBMC is dose
dependent.
FIG. 4 illustrates data demonstrating that the immunostimulatory
properties of siRNA are characteristic of a toll-like receptor
mediated immune response and that dendritic cells are one cell type
responsible for the IFN-.alpha. response to lipid-encapsulated
siRNA. FIG. 4A illustrates data demonstrating that the IFN-.alpha.
response to lipid-encapsulated siRNA is inhibited by chloroquine.
FIG. 4B illustrates data demonstrating that the IL-6 response to
lipid-encapsulated siRNA is inhibited by chloroquine. FIG. 4C
illustrates data demonstrating that dendritic cells are the primary
cell type responsible for the IFN-.alpha. response to
lipid-encapsulated siRNA.
FIG. 5 illustrates data demonstrating that immune stimulation by
siRNA duplexes is induced by a GU-rich motif (e.g., 5'-UGU-3' and
5'-UGUGU-3' motifs).
FIG. 6 illustrates data demonstrating that immune stimulation by
siRNA duplexes is induced by a GU-rich motif (e.g., 5'-UGU-3' and
5'-UGUGU-3' motifs).
FIG. 7 illustrates data demonstrating that immune stimulation by
siRNA duplexes is induced by a GU-rich motif (e.g., 5'-UGU-3' and
5'-UGUGU-3' motifs).
FIG. 8 illustrates data demonstrating that immune stimulation by
siRNA duplexes is induced by a GU-rich motif (e.g., 5'-UGU-3' and
5'-UGUGU-3' motifs).
FIG. 9 illustrates data demonstrating that plasma derived factors
enhance the immunostimulatory effects of siRNA in vitro and that
stimulation of human PBMC by siRNA was also dependent on nucleotide
sequence. FIG. 9A shows the immunostimulatory effects of .beta.gal
siRNA duplexes. FIG. 9B shows the immunostimulatory effects of BP1
siRNA duplexes.
FIG. 10 is Table 1 which summarizes data from in vitro and in vivo
experiments to measure the immunostimulatory effects of selected
siRNA molecules (SEQ ID NO:1-69).
FIG. 11 illustrates data demonstrating sequence dependent induction
of cytokines by systemically administered siRNA. FIG. 11A
illustrates serum IFN-.alpha. levels 6 h after intravenous
administration of 50 .mu.g (.about.2 mg/kg) encapsulated siRNA
targeting luciferase (Luc), .beta.-galactosidase (.beta.-gal), BP1
or the respective non-targeting sequence control siRNA into ICR
mice. Injection of empty liposomes or naked .beta.-gal siRNA alone
induced no detectable IFN-.alpha.. FIG. 11B illustrates a dose
response to encapsulated .beta.-gal 728 siRNA measuring serum
IFN-.alpha. at 6 h. FIG. 11C illustrates data demonstrating serum
IFN-.alpha. levels 6 h after intravenous administration of 50 .mu.g
.beta.-gal 728, .beta.-gal 481, TetR 57 or TetR control siRNA
encapsulated in liposomes comprising DLinDMA in the lipid bilayer.
FIG. 11D illustrates data demonstrating that TNF-.alpha., IL-6 and
IFN-.gamma. are also induced by stimulatory siRNA.
FIG. 12 illustrates data demonstrating that the immune stimulatory
activity of siRNA is regulated by GU-rich motifs. FIG. 12A is Table
2 which sets forth the modified siRNA sequences (SEQ ID NOS:70-81)
used in this series of experiments. Series 1; .beta.-gal control
(highly stimulatory), .beta.-gal Mod1 (single base substitution)
and .beta.-gal Mod2 (double base substitution). Series 2; BP1
control (low stimulatory), BP1 Mod1 (single base substitution) and
BP1 Mod2 (double base substitution). Base substitutions are
underlined. FIG. 12B illustrates data demonstrating that siRNA can
be rendered more or less stimulatory by the introduction or
disruption of a 5'-UGUGU-3' motif respectively. FIG. 12C
illustrates data demonstrating that BP-1 siRNA modified to
incorporate GU-rich motifs have enhanced immune stimulatory
activity. FIG. 12D illustrates data demonstrating that .beta.-gal
siRNA modified to delete GU-rich motifs have reduced immune
stimulatory activity. FIG. 12E illustrates data demonstrating that
there is a drop in peripheral white blood cell and platelet counts
associated with administration of immunostimulatory siRNA and this
is ameliorated by RNA sequence modifications. FIG. 12F illustrates
data demonstrating that there is a drop in peripheral white blood
cell and platelet counts associated with administration of
immunostimulatory modified BP-1 siRNA.
FIG. 13 illustrates data demonstrating that lipid encapsulated
siRNA is effective at mediating RNAi in vitro.
FIG. 14 illustrates data demonstrating that freshly isolated
monocytes can be stimulated with lipid-complexed siRNA or
polycation-complexed siRNA to produce inflammatory cytokines. FIG.
14A illustrates data showing levels of TNF-.alpha. produced in
response to lipid-siRNA complexes. FIG. 14B illustrates data
showing levels of IL-6 produced in response to lipid-siRNA
complexes. FIG. 14C illustrates data showing levels of IFN-.alpha.,
IL-6, and TNF-.alpha. produced in response to polycation-siRNA
complexes.
FIG. 15 illustrates data demonstrating that the immunostimulatory
activity of siRNA is not caused by contaminants such as ssRNA. FIG.
15A illustrates data demonstrating that PAGE purification of the
siRNA duplex does not affect its immunostimulatory activity. FIG.
15B illustrates data demonstrating that GU rich sense ssRNA induced
no detectable IFN-.alpha. following RNase A treatment and that
RNase A treatment had minimal effect on the induction of
IFN-.alpha. by siRNA duplex compared to untreated samples.
FIG. 16 is Table 3 which sets forth certain siRNA sequences (SEQ ID
NOS: 82-93) used in the experiments described herein.
FIG. 17 illustrates data demonstrating that siRNA can be designed
that are active in mediating RNAi and have minimal capacity to
activate innate immune responses. FIG. 17A is Table 4 which sets
forth siRNA sequences (SEQ ID NOS: 94-101) designed to target
.beta.-gal (codon start sites 478, 924, and 2891) that lack
putative immunostimulatory motifs. FIG. 17B illustrates data
demonstrating the immunostimulatory activity of novel.beta.-gal
siRNA on human PBMC. FIG. 17C illustrates data demonstrating
inhibition of.beta.-gal activity by novel .beta.-gal targeting
siRNA in Neuro 2A cells. FIG. 17D illustrates data demonstrating
inhibition of .beta.-gal activity by GU-rich.beta.-gal targeting
siRNA in Neuro2A cells.
FIG. 18 illustrates data demonstrating that the cytokine response
to siRNA in vivo is not limited to pDC cells.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
The present invention is based, in part, on the surprising
discovery that siRNA molecules have immunostimulatory effects that
can be modulated. In particular, the invention is based on the
discovery that siRNA molecules comprising GU-rich motifs (e.g.,
siRNA molecules comprising, a 5'-GU-3' motif, a 5'-UG-3' motif, a
5'-UGU-3' motif, a 5'-GUGU-3' motif, or a 5'-UGUGU-3' motif) have
immunostimulatory properties. Based on this discovery, the
invention provides methods and compositions for enhancing or
decreasing the immune response associated with siRNA molecules as
well as methods for identifying siRNA molecules with
immunostimulatory properties or non-immunostimulatory
properties.
For example the immunostimulatory properties of an siRNA molecule
comprising a GU-rich motif can be decreased by substituting one
more of the G's or one or more of the U's with another nucleotide.
Likewise, the immunostimulatory properties of an siRNA molecule can
be increased by a substitution or substitutions that introduce a
GU-rich motif into the siRNA sequence. In addition, the
immunostimulatory properties of an siRNA molecule comprising a
GU-rich motif can be increased by a substitution or substitutions
that introduce further GU-rich motifs into the sequence.
Alternatively, an siRNA that is not immunostimulatory may be
modified so that it is immunostimulatory by a substitution that
introduces a GU-rich motif into the siRNA sequence.
Without being bound by theory, it is postulated that the siRNA
molecules' immunostimulatory activity is mediated by Toll-Like
Receptor mediated signaling. These findings have significant
implications for the clinical development of RNAi as a novel
therapeutic approach and in the interpretation of specific gene
silencing effects using siRNA. For example, immunostimulatory
siRNAs can be modified to disrupt a GU-rich motif, thus reducing
their immunostimulatory properties while retaining their ability to
silence a target gene. Alternatively, the immunostimulatory siRNAs
can be used to generate controlled, transient cytokine production,
activated T cell and NK cell proliferation, tumor-specific CTL
responses, non-gene specific tumor regression, and B cell
activation (i.e., antibody production). In addition,
non-immunostimulatory siRNAs can be modified to comprise a GU-rich
motif, thus enhancing their immunostimulatory properties while
retaining their ability to silence a target gene.
II. Definitions
The term "interfering RNA" or "RNAi" or "interfering RNA sequence"
refers to double-stranded RNA (i.e., duplex RNA) that is capable of
reducing or inhibiting expression of a target gene (i.e., by
mediating the degradation of mRNAs which are complementary to the
sequence of the interfering RNA) when the interfering RNA is in the
same cell as the target gene. Interfering RNA thus refers to the
double stranded RNA formed by two complementary strands or by a
single, self-complementary strand. Interfering RNA may have has
substantial or complete identity to the target gene or may comprise
a region of mismatch (i.e., a mismatch motif). The sequence of the
interfering RNA can correspond to the full length target gene, or a
subsequence thereof. Interfering RNA includes small-interfering
RNA" or "siRNA," i.e., interfering RNA of about 15-60, 15-50,
15-50, or 15-40 (duplex) nucleotides in length, more typically
about, 15-30, 15-25 or 19-25 (duplex) nucleotides in length, and is
preferably about 20-24 or about 21-22 or 21-23 (duplex) nucleotides
in length (e.g., each complementary sequence of the double stranded
siRNA is 15-60, 15-50, 15-50, 15-40, 15-30, 15-25 or 19-25
nucleotides in length, preferably about 20-24 or about 21-22 or
21-23 nucleotides in length, and the double stranded siRNA is about
15-60, 15-50, 15-50, 15-40, 15-30, 15-25 or 19-25 preferably about
20-24 or about 21-22 or 21-23 base pairs in length). siRNA duplexes
may comprise 3' overhangs of about 1 to about 4 nucleotides,
preferably of about 2 to about 3 nucleotides and 5' phosphate
termini. The siRNA can be chemically synthesized or may be encoded
by a plasmid (e.g., transcribed as sequences that automatically
fold into duplexes with hairpin loops). siRNA can also be generated
by cleavage of longer dsRNA (e.g., dsRNA greater than about 25
nucleotides in length) with the E coli RNase III or Dicer. These
enzymes process the dsRNA into biologically active siRNA (see,
e.g., Yang et al., PNAS USA 99: 9942-7 (2002); Calegari et al.,
PNAS USA 99: 14236 (2002); Byrom et al., Ambion TechNotes 10(1):
4-6 (2003); Kawasaki et al., Nucleic Acids Res. 31: 981-7 (2003);
Knight and Bass, Science 293: 2269-71 (2001); and Robertson et al.,
J. Biol. Chem. 243: 82 (1968)). Preferably, dsRNA are at least 50
nucleotides to about 100, 200, 300, 400 or 500 nucleotides in
length. A dsRNA may be as long as 1000, 1500, 2000, 5000
nucleotides in length, or longer. The dsRNA can encode for an
entire gene transcript or a partial gene transcript.
As used herein, the term "mismatch motif" or "mismatch region"
refers to a portion of an siRNA sequence that does not have 100%
complementarity to its target sequence. An siRNA may have at least
one, two, three, four, five, six, or more mismatch regions. The
mismatch regions may be contiguous or may be separated by 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides. The mismatch
motifs or regions may comprise a single nucleotide or may comprise
two, three, four, five, or more nucleotides. The mismatch motifs or
regions may be immunostimulatory or non-immunostimulatory. An
immunostimulatory mismatch motif or region is a GU-rich motif
(e.g., 5'-GU-3' motif, a 5'-UG-3' motif, a 5'-UGU-3' motif, a
5'-GUGU-3' motif, or a 5'-UGUGU-3' motif). A non-immunostimulatory
mismatch motif or region lacks a GU-rich motif.
An "effective amount" or "therapeutically effective amount" of an
siRNA is an amount sufficient to produce the desired effect, e.g.,
a inhibition of expression of a target sequence in comparison to
the normal expression level detected in the absence of the siRNA,
or e.g., an increase or decrease in the immune response in
comparison to the normal level detected in the absence of the
siRNA. Inhibition of expression of a target gene or target sequence
is achieved when the value obtained with the construct relative to
the control is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%,
15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a
target gene or target sequence include, e.g., examination of
protein or mRNA levels using techniques known to those of skill in
the art such as dot blots, northern blots, in situ hybridization,
ELISA, immunoprecipitation, enzyme function, as well as phenotypic
assays known to those of skill in the art.
By "increase" or "increasing" of an immune response by an siRNA is
intended to mean a detectable increase of an immune response to the
siRNA (e.g., a modified or unmodified siRNA comprising a GU-rich
motif). The amount of increase of an immune response by of a
modified or unmodified siRNA comprising a GU-rich motif may be
determined relative to the level of an immune response that is
detected in the absence of the siRNA. The amount of increase of an
immune response by a modified siRNA comprising a GU-rich motif may
also be determined relative to the level of an immune response in
the presence of an unmodified siRNA (e.g., an unmodified siRNA
lacking a GU-rich motif). A detectable increase can be about 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more higher
than the immune response detected in the absence of the siRNA
(e.g., a modified or unmodified siRNA comprising a GU-rich motif).
An increase in the immune response to siRNA is typically measured
by an increase in cytokine production (e.g., IFN.gamma.,
IFN.alpha., TNF.alpha., IL-6, or IL-12) by a responder cell in
vitro or an increase in cytokine production in the sera of a
mammalian subject after administration of the siRNA.
By "decrease" or "decreasing" of an immune response by an siRNA is
intended to mean a detectable decrease of an immune response to
siRNA (e.g., a modified siRNA lacking a GU-rich motif). The amount
of decrease of an immune response by a modified siRNA lacking a
GU-rich motif may be determined relative to the level of an immune
response in the presence of an unmodified siRNA (e.g., an
unmodified siRNA comprising a GU-rich motif). A detectable decrease
can be about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%
or more lower than the immune response detected in the absence of
the unmodified siRNA (e.g., an unmodified siRNA comprising a
GU-rich motif). An increase in the immune response to siRNA is
typically measured by an increase in cytokine production (e.g.,
IFN.gamma., IFN.alpha., TNF.alpha., IL-6, or IL-12) by a responder
cell in vitro or an increase in cytokine production in the sera of
a mammalian subject after administration of the siRNA.
As used herein, the term "responder cell" refers to a cell,
preferable a mammalian cell, that produces a detectable immune
response when contacted with an immunostimulatory double stranded
RNA. Exemplary responder cells include, e.g., dendritic cells,
macrophages, peripheral blood mononuclear cells, splenocytes, and
the like. Detectable immune responses include, e.g., production of
cytokines such as IFN-.alpha., IFN-.gamma., TNF-.alpha., IL-1,
IL-2, IL-3, 11-4, IL-5, IL-6, IL-10, IL-12, IL-13, and TGF.
"Substantial identity" refers to a sequence that hybridizes to a
reference sequence under stringent conditions, or to a sequence
that has a specified percent identity over a specified region of a
reference sequence.
The phrase "stringent hybridization conditions" refers to
conditions under which an siRNA will hybridize to its target
sequence, typically in a complex mixture of nucleic acids, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization.
Exemplary stringent hybridization conditions can be as following:
50% formamide, 5.times.SSC, and 1% SDS, incubating at 42.degree.
C., or, 5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash
in 0.2.times.SSC, and 0.1% SDS at 65.degree. C. For PCR, a
temperature of about 36.degree. C. is typical for low stringency
amplification, although annealing temperatures may vary between
about 32.degree. C. and 48.degree. C. depending on primer length.
For high stringency PCR amplification, a temperature of about
62.degree. C. is typical, although high stringency annealing
temperatures can range from about 50.degree. C. to about 65.degree.
C., depending on the primer length and specificity. Typical cycle
conditions for both high and low stringency amplifications include
a denaturation phase of 90.degree. C.-95.degree. C. for 30 sec-2
min., an annealing phase lasting 30 sec.-2 min., and an extension
phase of about 72.degree. C. for 1-2 min. Protocols and guidelines
for low and high stringency amplification reactions are provided,
e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and
Applications, Academic Press, Inc. New York).
Nucleic acids that do not hybridize to each other under stringent
conditions are still substantially identical if the polypeptides
which they encode are substantially identical. This occurs, for
example, when a copy of a nucleic acid is created using the maximum
codon degeneracy permitted by the genetic code. In such cases, the
nucleic acids typically hybridize under moderately stringent
hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
reference, e.g., and Current Protocols in Molecular Biology, ed.
Ausubel, et al.
The terms "substantially identical" or "substantial identity," in
the context of two or more nucleic acids, refer to two or more
sequences or subsequences that are the same or have a specified
percentage of nucleotides that are the same (i.e., at least about
60%, preferably 65%, 70%, 75%, preferably 80%, 85%, 90%, or 95%
identity over a specified region), when compared and aligned for
maximum correspondence over a comparison window, or designated
region as measured using one of the following sequence comparison
algorithms or by manual alignment and visual inspection. This
definition, when the context indicates, also refers analogously to
the complement of a sequence. Preferably, the substantial identity
exists over a region that is at least about 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 75, or 100 nucleotides in length.
For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a
sequence comparison algorithm, test and reference sequences are
entered into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. Default program parameters can be used, or alternative
parameters can be designated. The sequence comparison algorithm
then calculates the percent sequence identities for the test
sequences relative to the reference sequence, based on the program
parameters.
A "comparison window", as used herein, includes reference to a
segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
A preferred example of algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al., Nuc.
Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.
215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used,
with the parameters described herein, to determine percent sequence
identity for the nucleic acids and proteins of the invention.
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information.
The BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.2, more preferably less than about 0.01, and
most preferably less than about 0.001.
The term "nucleic acid" or "polynucleotide" refers to a polymer
containing at least two deoxyribonucleotides or ribonucleotides in
either single- or double-stranded form and include DNA and RNA. DNA
may be in the form of, e.g., antisense oligonucleotides, plasmid
DNA, pre-condensed DNA, a PCR product, vectors (P1, PAC, BAC, YAC,
artificial chromosomes), expression cassettes, chimeric sequences,
chromosomal DNA, or derivatives and combinations of these groups.
RNA may be in the form of siRNA, mRNA, tRNA, rRNA, tRNA, vRNA, and
combinations thereof. Unless specifically limited, the terms
encompasses nucleic acids containing known analogues of natural
nucleotides that have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g., degenerate codon substitutions),
alleles, orthologs, SNPs, and complementary sequences as well as
the sequence explicitly indicated. Specifically, degenerate codon
substitutions may be achieved by generating sequences in which the
third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem. 260:2605-2608 (1985); and Cassol et al. (1992); Rossolini et
al., Mol. Cell. Probes 8:91-98 (1994)). "Nucleotides" contain a
sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate
group. Nucleotides are linked together through the phosphate
groups. "Bases" include purines and pyrimidines, which further
include natural compounds adenine, thymine, guanine, cytosine,
uracil, inosine, and natural analogs, and synthetic derivatives of
purines and pyrimidines, which include, but are not limited to,
modifications which place new reactive groups such as, but not
limited to, amines, alcohols, thiols, carboxylates, and
alkylhalides.
The term "gene" refers to a nucleic acid (e.g., DNA or RNA)
sequence that comprises partial length or entire length coding
sequences necessary for the production of a polypeptide or
precursor polypeptide.
"Gene product," as used herein, refers to a product of a gene such
as an RNA transcript or a polypeptide.
The term "lipid" refers to a group of organic compounds that
include, but are not limited to, esters of fatty acids and are
characterized by being insoluble in water, but soluble in many
organic solvents. They are usually divided into at least three
classes: (1) "simple lipids which include fats and oils as well as
waxes; (2) "compound lipids" which include phospholipids and
glycolipids; (3) "derived lipids" such as steroids.
"Lipid vesicle" refers to any lipid composition that can be used to
deliver a compound including, but not limited to, liposomes,
wherein an aqueous volume is encapsulated by an amphipathic lipid
bilayer; or wherein the lipids coat an interior comprising a large
molecular component, such as a plasmid comprising an interfering
RNA sequence, with a reduced aqueous interior; or lipid aggregates
or micelles, wherein the encapsulated component is contained within
a relatively disordered lipid mixture.
As used herein, "lipid encapsulated" can refer to a lipid
formulation that provides a compound, such as siRNA, with full
encapsulation, partial encapsulation, or both. In a preferred
embodiment, the nucleic acid is fully encapsulated in the lipid
formulation (e.g., to form an SPLP, pSPLP, SNALP, or other nucleic
acid-lipid particle).
The nucleic acid-lipid particles of the present invention typically
have a mean diameter of about 50 nm to about 150 nm, more typically
about 100 nm to about 130 nm, most typically about 110 nm to about
115 nm, and are substantially nontoxic. In addition, the nucleic
acids when present in the nucleic acid-lipid particles of the
present invention are resistant in aqueous solution to degradation
with a nuclease. Nucleic acid-lipid particles and their method of
preparation are disclosed in U.S. Pat. Nos. 5,976,567; 5,981,501;
6,534,484; 6,586,410; 6,815,432; and WO 96/40964.
Various suitable cationic lipids may be used in the present
invention, either alone or in combination with one or more other
cationic lipid species or non-cationic lipid species.
The cationic lipids described herein typically carry a net positive
charge at a selected pH, such as physiological pH. It has been
surprisingly found that cationic lipids comprising alkyl chains
with multiple sites of unsaturation, e.g., at least two or three
sites of unsaturation, are particularly useful for forming nucleic
acid-lipid particles with increased membrane fluidity. A number of
cationic lipids and related analogs, which are also useful in the
present invention, have been described in U.S. Patent Application
Nos. 60/578,075 and 60/610,746; U.S. Pat. Nos. 5,753,613;
5,208,036, 5,264,618, 5,279,833 and 5,283,185, and WO 96/10390.
The non-cationic lipids used in the present invention can be any of
a variety of neutral uncharged, zwitterionic or anionic lipids
capable of producing a stable complex. They are preferably neutral,
although they can alternatively be positively or negatively
charged. Examples of non-cationic lipids useful in the present
invention include: phospholipid-related materials, such as
lecithin, phosphatidylethanolamine, lysolecithin,
lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cephalin, cardiolipin,
phosphatidic acid, cerebrosides, dicetylphosphate,
phospholipid-related materials, such as lecithin,
phosphatidylethanolamine, lysolecithin,
lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cephalin, cardiolipin,
phosphatidic acid, cerebrosides, dicetylphosphate,
distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE). Non-cationic
lipids or sterols such as cholesterol may be present. Additional
nonphosphorous containing lipids are, e.g., stearylamine,
dodecylamine, hexadecylamine, acetyl palmitate,
glycerolricinoleate, hexadecyl stereate, isopropyl myristate,
amphoteric acrylic polymers, triethanolamine-lauryl sulfate,
alkyl-aryl sulfate polyethyloxylated fatty acid amides,
dioctadecyldimethyl ammonium bromide and the like,
diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, and cerebrosides. Other lipids
such as lysophosphatidylcholine and lysophosphatidylethanolamine
may be present. Non-cationic lipids also include polyethylene
glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene
glycol conjugated to phospholipids or to ceramides (referred to as
PEG-Cer), as described in U.S. Pat. No. 5,820,873.
In preferred embodiments, the non-cationic lipids are
diacylphosphatidylcholine (e.g., distearoylphosphatidylcholine,
dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine and
dilinoleoylphosphatidylcholine), diacylphosphatidylethanolamine
(e.g., dioleoylphosphatidylethanolamine and
palmitoyloleoylphosphatidylethanolamine), ceramide or
sphingomyelin. The acyl groups in these lipids are preferably acyl
groups derived from fatty acids having C.sub.10-C.sub.24 carbon
chains. More preferably the acyl groups are lauroyl, myristoyl,
palmitoyl, stearoyl or oleoyl. In particularly preferred
embodiments, the non-cationic lipid will be cholesterol,
1,2-sn-dioleoylphosphatidylethanolamine, or egg sphingomyelin
(ESM).
In addition to cationic and non-cationic lipids, the SPLPs of the
present invention comprise bilayer stabilizing component (BSC) such
as an ATTA-lipid or a PEG-lipid, such as PEG coupled to
dialkyloxypropyls (PEG-DAA) (see, copending U.S. patent application
Ser. No. 10/942,379), PEG coupled to diacylglycerol (PEG-DAG) (see,
copending U.S. patent application Ser. No. 10/136,707), PEG coupled
to phosphatidylethanolamine (PE) (PEG-PE) or some other
phospholipid, or PEG conjugated to ceramides (PEG-Cer), or a
mixture thereof (see, U.S. Pat. No. 5,885,613). In one preferred
embodiment, the BSC is a conjugated lipid that inhibits aggregation
of the SPLPs. Suitable conjugated lipids include, but are not
limited to, PEG-lipid conjugates, ATTA-lipid conjugates,
cationic-polymer-lipid conjugates (CPLs) or mixtures thereof. In
one preferred embodiment, the SPLPs comprise either a PEG-lipid
conjugate or an ATTA-lipid conjugate together with a CPL.
PEG is a polyethylene glycol, a linear, water-soluble polymer of
ethylene PEG repeating units with two terminal hydroxyl groups.
PEGs are classified by their molecular weights; for example, PEG
2000 has an average molecular weight of about 2,000 daltons, and
PEG 5000 has an average molecular weight of about 5,000 daltons.
PEGs are commercially available from Sigma Chemical Co. as well as
other companies and include, for example, the following:
monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene
glycol-succinate (MePEG-S), monomethoxypolyethylene
glycol-succinimidyl succinate (MePEG-S--NHS),
monomethoxypolyethylene glycol-amine (MePEG-NH.sub.2),
monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and
monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). In
addition, monomethoxypolyethyleneglycol-acetic acid
(MePEG-CH.sub.2COOH), is particularly useful for preparing the
PEG-lipid conjugates including, e.g., PEG-DAA conjugates.
In a preferred embodiment, the PEG has an average molecular weight
of from about 1000 to about 5000 daltons, more preferably, from
about 1,000 to about 3,000 daltons and, even more preferably, of
about 2,000 daltons. The PEG can be optionally substituted by an
alkyl, alkoxy, acyl or aryl group. PEG can be conjugated directly
to the lipid or may be linked to the lipid via a linker moiety. Any
linker moiety suitable for coupling the PEG to a lipid can be used
including, e.g., non-ester containing linker moieties and
ester-containing linker moieties.
As used herein, the term "non-ester containing linker moiety"
refers to a linker moiety that does not contain a carboxylic ester
bond (--OC(O)--). Suitable non-ester containing linker moieties
include, but are not limited to, amido (--C(O)NH--), amino
(--NR--), carbonyl (--C(O)--), carbamate (--NHC(O)O--), urea
(--NHC(O)NH--), disulphide (--S--S--), ether (--O--), succinyl
(--(O)CCH.sub.2CH.sub.2C(O)--), succinamidyl
(--NHC(O)CH.sub.2CH.sub.2C(O)NH--), ether, disulphide, etc. as well
as combinations thereof (such as a linker containing both a
carbamate linker moiety and an amido linker moiety). In a preferred
embodiment, a carbamate linker is used to couple the PEG to the
lipid.
In other embodiments, an ester containing linker moiety is used to
couple the PEG to the lipid. Suitable ester containing linker
moieties include, e.g., carbonate (--OC(O)O--), succinoyl,
phosphate esters (--O--(O)POH--O--), sulfonate esters, and
combinations thereof.
As used herein, the term "SNALP" refers to a stable nucleic
acid-lipid particle, including SPLP. A SNALP represents a lipid
vesicle oencapsulating a nucleic acid (e.g., ssDNA, dsDNA, ssRNA,
dsRNA, siRNA, or a plasmid, including plasmids from which an
interfering RNA is transcribed). As used herein, the term "SPLP"
refers to a nucleic acid-lipid particle comprising a nucleic acid
(e.g., a plasmid) encapsulated within a lipid vesicle. SNALPs and
SPLPs typically contain a cationic lipid, a non-cationic lipid, and
a lipid that prevents aggregation of the particle (e.g., a
PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for
systemic applications, as they exhibit extended circulation
lifetimes following intravenous (i.v.) injection, accumulate at
distal sites (e.g., sites physically separated from the
administration site) and can mediate expression of the transfected
gene at these distal sites. SPLPs include "pSPLP" which comprise an
encapsulated condensing agent-nucleic acid complex as set forth in
WO 00/03683.
The term "vesicle-forming lipid" is intended to include any
amphipathic lipid having a hydrophobic moiety and a polar head
group, and which by itself can form spontaneously into bilayer
vesicles in water, as exemplified by most phospholipids.
The term "vesicle-adopting lipid" is intended to include any
amphipathic lipid that is stably incorporated into lipid bilayers
in combination with other amphipathic lipids, with its hydrophobic
moiety in contact with the interior, hydrophobic region of the
bilayer membrane, and its polar head group moiety oriented toward
the exterior, polar surface of the membrane. Vesicle-adopting
lipids include lipids that on their own tend to adopt a nonlamellar
phase, yet which are capable of assuming a bilayer structure in the
presence of a bilayer-stabilizing component. A typical example is
DOPE (dioleoylphosphatidylethanolamine). Bilayer stabilizing
components include, but are not limited to, conjugated lipids that
inhibit aggregation of nucleic acid-lipid particles, polyamide
oligomers (e.g., ATTA-lipid derivatives), peptides, proteins,
detergents, lipid-derivatives, PEG-lipid derivatives such as PEG
coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG
coupled to phosphatidyl-ethanolamines, PEG conjugated to ceramides
(see, U.S. Pat. No. 5,885,613); cationic PEG lipids, and mixture
thereof.
The term "amphipathic lipid" refers, in part, to any suitable
material wherein the hydrophobic portion of the lipid material
orients into a hydrophobic phase, while the hydrophilic portion
orients toward the aqueous phase. Amphipathic lipids are usually
the major component of a lipid vesicle. Hydrophilic characteristics
derive from the presence of polar or charged groups such as
carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl,
nitro, hydroxy and other like groups. Hydrophobicity can be
conferred by the inclusion of apolar groups that include, but are
not limited to, long chain saturated and unsaturated aliphatic
hydrocarbon groups and such groups substituted by one or more
aromatic, cycloaliphatic or heterocyclic group(s). Examples of
amphipathic compounds include, but are not limited to,
phospholipids, aminolipids and sphingolipids. Representative
examples of phospholipids include, but are not limited to,
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidic acid, palmitoyloleoyl
phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine or
dilinoleoylphosphatidylcholine. Other compounds lacking in
phosphorus, such as sphingolipid, glycosphingolipid families,
diacylglycerols and .beta.-acyloxyacids, are also within the group
designated as amphipathic lipids. Additionally, the amphipathic
lipid described above can be mixed with other lipids including
triglycerides and sterols.
The term "neutral lipid" refers to any of a number of lipid species
that exist either in an uncharged or neutral zwitterionic form at a
selected pH. At physiological pH, such lipids include, for example,
diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and
diacylglycerols.
The term "non-cationic lipid" refers to any neutral lipid as
described above as well as anionic lipids.
The term "anionic lipid" refers to any lipid that is negatively
charged at physiological pH. These lipids include, but are not
limited to, phosphatidylglycerol, cardiolipin,
diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl
phosphatidylethanolamines, N-succinyl phosphatidylethanolamines,
N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,
palmitoyloleyolphosphatidylglycerol (POPG), and other anionic
modifying groups joined to neutral lipids.
The term "cationic lipid" refers to any of a number of lipid
species that carry a net positive charge at a selected pH, such as
physiological pH. Such lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP"); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
("DC-Chol");
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"); 1,2-DiLinoleyloxy-N,N-dimethylaminopropane
(DLinDMA); and 1,2-Dilinolenyloxy-N,N-dimethylaminopropane
(DLenDMA). The following lipids are cationic and have a positive
charge at below physiological pH: DODAP, DODMA, DMDMA and the
like.
The term "hydrophobic lipid" refers to compounds having apolar
groups that include, but are not limited to, long chain saturated
and unsaturated aliphatic hydrocarbon groups and such groups
optionally substituted by one or more aromatic, cycloaliphatic or
heterocyclic group(s). Suitable examples include, but are not
limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino,
1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.
The term "fusogenic" refers to the ability of a liposome, an SNALP
or other drug delivery system to fuse with membranes of a cell. The
membranes can be either the plasma membrane or membranes
surrounding organelles, e.g., endosome, nucleus, etc.
The term "diacylglycerol" refers to a compound having 2-fatty acyl
chains, R.sup.1 and R.sup.2, both of which have independently
between 2 and 30 carbons bonded to the 1- and 2-position of
glycerol by ester linkages. The acyl groups can be saturated or
have varying degrees of unsaturation. Diacylglycerols have the
following general formula:
##STR00001##
The tenn "dialkyloxypropyl" refers to a compound having 2-alkyl
chains, R.sup.1 and R.sup.2, both of which have independently
between 2 and 30 carbons. The alkyl groups can be saturated or have
varying degrees of unsaturation. Dialkyloxypropyls have the
following general formula:
##STR00002##
The term "ATTA" or "polyamide" refers to, but is not limited to,
compounds disclosed in U.S. Pat. Nos. 6,320,017 and 6,586,559.
These compounds include a compound having the formula
##STR00003## wherein: R is a member selected from the group
consisting of hydrogen, alkyl and acyl; R.sup.1 is a member
selected from the group consisting of hydrogen and alkyl; or
optionally, R and R.sup.1 and the nitrogen to which they are bound
form an azido moiety; R.sup.2 is a member of the group selected
from hydrogen, optionally substituted alkyl, optionally substituted
aryl and a side chain of an amino acid; R.sup.3 is a member
selected from the group consisting of hydrogen, halogen, hydroxy,
alkoxy, mercapto, hydrazino, amino and NR.sup.4R.sup.5, wherein
R.sup.4 and R.sup.5 are independently hydrogen or alkyl; n is 4 to
80; m is 2 to 6; p is 1 to 4; and q is 0 or 1. It will be apparent
to those of skill in the art that other polyamides can be used in
the compounds of the present invention.
As used herein, the term "aqueous solution" refers to a composition
comprising in whole, or in part, water.
As used herein, the term "organic lipid solution" refers to a
composition comprising in whole, or in part, an organic solvent
having a lipid.
"Distal site," as used herein, refers to a physically separated
site, which is not limited to an adjacent capillary bed, but
includes sites broadly distributed throughout an organism.
"Serum-stable" in relation to nucleic acid-lipid particles means
that the particle is not significantly degraded after exposure to a
serum or nuclease assay that would significantly degrade free DNA.
Suitable assays include, for example, a standard serum assay or a
DNAse assay such as those described in the Examples below.
"Systemic delivery," as used herein, refers to delivery that leads
to a broad biodistribution of a compound within an organism. Some
techniques of administration can lead to the systemic delivery of
certain compounds, but not others. Systemic delivery means that a
useful, preferably therapeutic, amount of a compound is exposed to
most parts of the body. To obtain broad biodistribution generally
requires a blood lifetime such that the compound is not rapidly
degraded or cleared (such as by first pass organs (liver, lung,
etc.) or by rapid, nonspecific cell binding) before reaching a
disease site distal to the site of administration. Systemic
delivery of nucleic acid-lipid particules can be by any means known
in the art including, for example, intravenous, subcutaneous,
intraperitoneal, In a preferred embodiment, systemic delivery of
nucleic acid-lipid particles is by intravenous delivery.
"Local delivery," as used herein, refers to delivery of a compound
directly to a target site within an organism. For example, a
compound can be locally delivered by direct injection into a
disease site such as a tumor or other target site such as a site of
inflammation or a target organ such as the liver, heart, pancreas,
kidney, and the like.
III. siRNAs
The siRNA of the invention are capable of silencing expression of a
target sequence, are about 15 to 30 nucleotides in length, and
comprise at least one mismatch motif relative to a target nucleic
acid sequence. The mismatch motif may be immunostimulatory (i.e.,
GU-rich) or may be non-immunostumulatory. The siRNAs that are
immunostimulatory comprise one or more GU-rich motifs (e.g, a
5'-GU-3' motif, a 5'-UG-3' motif, a 5'-UGU-3' motif, a 5'-GUGU-3'
motif, or a 5'-UGUGU-3' motif). The siRNA which are not
immunostimulatory or are less immunostimulatory may comprise one
GU-rich motif, but will typically not comprise such motifs. The
siRNA sequences may have overhangs (e.g., 3' or 5' overhangs as
described in (Elbashir, et al., Genes Dev. 15:188 (2001); Nykanen,
et al., Cell 107:309 (2001)) or may lack overhangs (i.e., have
blunt ends).
According to the methods of the invention, siRNA which are
immunostimulatory can be modified to decrease their
immunostimulatory properties. For example, an immunostimulatory
siRNA comprising a GU-rich motif can be modified to disrupt or
eliminate the motif, i.e., by replacing one or more of the G's or
one or more of the U's in the GU-rich motif with another
nucleotide, thus generating an siRNA with reduced immunostimulatory
properties. Alternatively, siRNA which are not immunostimulatory
can be modified to add a GU-rich motif, i.e., by substitution of a
nucleotide with a G or a U, thus generating an siRNA with enhanced
immunostimulatory properties. In a preferred embodiment, siRNA
which are immunostimulatory are modified to decrease their
immunostimulatory properties, e.g., to disrupt a GU-rich motif.
The siRNA molecules described herein typically comprise at least
one mismatch region (e.g., an immunostimulatory mismatch region or
a non-immunostimulatory mismatch region) with its target sequence.
An siRNA molecule is modified to either enhance its
immunostimulatory properties or to decrease its immunostimulatory
properties. For example, an siRNA molecule modified to reduce its
immunostimulatory properties is typically modified to comprise at
least one non-immunostimulatory mismatch region relative to its
target sequence. In contrast, an siRNA modified to enhance its
immunostimulatory properties is typically modified to comprise at
least one immunostimulatory mismatch region relative to its target
sequence.
A. Selection of siRNA sequences
Suitable siRNA sequences can be identified using any means known in
the art. Typically, the methods described in Elbashir, et al.,
Nature 411:494-498 (2001) and Elbashir, et al., EMBO J 20:
6877-6888 (2001) are combined with rational design rules set forth
in Reynolds et al., Nature Biotech. 22(3):326-330 (2004).
Typically, the sequence within about 50 to about 100 nucleotides 3'
of the AUG start codon of a transcript from the target gene of
interest is scanned for dinucleotide sequences (e.g., AA, CC, GG,
or UU) (see, e.g., Elbashir, et al., EMBO J 20: 6877-6888 (2001)).
The nucleotides immediately 3' to the dinucleotide sequences are
identified as potential siRNA target sequences. Typically, the 19,
21, 23, 25, 27, 29, 31, 33, 35 or more nucleotides immediately 3'
to the dinucleotide sequences are identified as potential siRNA
target sites. In some embodiments, the dinucleotide sequence is an
AA sequence and the 19 nucleotides immediately 3' to the AA
dinucleotide are identified as a potential siRNA target site.
Typically siRNA target sites are spaced at different postitions
along the length of the target gene. To further enhance silencing
efficiency of the siRNA sequences, potential siRNA target sites may
be further analyzed to identify sites that do not contain regions
of homology to other coding sequences. For example, a suitable
siRNA target site of about 21 base pairs typically will not have
more than 16-17 contiguous base pairs of homology to other coding
sequences. If the siRNA sequences are to be expressed from an RNA
Pol III promoter, siRNA target sequences lacking more than 4
contiguous A's or T's are selected.
Once the potential siRNA target site has been identified siRNA
sequences complementary to the siRNA target sites may be designed.
To enhance their silencing efficiency, the siRNA sequences may also
be analyzed by a rational design algorithm to identify sequences
that have one or more of the following features: (1) G/C content of
about 25% to about 60% G/C; (2) at least 3 A/Us at positions 15-19
of the sense strand; (3) no internal repeats; (4) an A at position
19 of the sense strand; (5) an A at position 3 of the sense strand;
(6) a U at position 10 of the sense strand; (7) no G/C at position
19 of the sense strand; and (8) no G at position 13 of the sense
strand. siRNA design tools that incorporate algorithms that assign
suitable values of each of these features are useful for selection
of siRNA.
Once a potential siRNA sequence has been identified, the sequence
can be analyzed for the presence of GU-rich motifs (e.g., 5'-GU-3',
5'-UGU-3', 5'-GUGU-3', or 5'-UGUGU-3' motifs. Potential siRNA
target sequences that contain GU-rich motifs are identified as
immunustimulatory siRNAs. Potential siRNA target sequences that
lack GU-rich motifs are identified as non-immunustimulatory siRNAs.
In some embodiments, potential siRNA target sequences comprising
GU-rich motifs are modified as described herein to eliminate the
motifs and reduce the immunostimulatory properties of the
sequences. In other embodiments, potential siRNA target sequences
lacking GU-rich motifs are modified as described herein to
introduce the motifs and increase the immunostimulatory properties
of the sequences. The immunostimulatory properties of the potential
siRNA target sequences can be confirmed using the assays described
in detail below.
One embodiment of the invention provides methods of identifying
siRNA molecules that are immunostimulatory or
non-immunostimulatory. Once identified, the immunostimulatory siRNA
molecules can be modified to increase or decrease their
immunostimulatory properties and the non-immunostimulatory
molecules can be modified so that they possess immunostimulatory
properties
In this embodiment, a target nucleic acid sequence is analyzed for
the presence of an immunostimulatory motif, e.g., a GU-rich motif.
If an immunostimulatory motif is present, a double stranded RNA
(i.e., siRNA) sequence having immunostimulatory properties is
identified. If no immunostimulatory motif is present, a double
stranded RNA (i.e., siRNA) sequence having non-immunostimulatory
properties is identified. The siRNA is then selected and contacted
with a mammalian responder cell under conditions such that the cell
produces a detectable immune response, thus confirming that the
siRNA is an immunostimulatory or a non-immunostimulatory siRNA. The
mammalian responder cell may be from a naive mammal (i.e., a mammal
that has not previously been in contact with the gene product of
the target nucleic acid sequence). The responder cell may be, e.g.,
a peripheral blood mononuclear cell, a macrophage, and the like.
The detectable immune response may comprise production of a
cytokine or growth factor such as, e.g., TNF-.alpha., IFN-.alpha.,
IL-6, IL-12, and combinations thereof.
Suitable assays to detect an immune response induced by
immunostimulatory siRNA include the double monoclonal antibody
sandwich immunoassay technique of David et al. U.S. Pat. No.
4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et
al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and
S. Livingstone, Edinburgh (1970)); the "western blot" method of
Gordon et al. (U.S. Pat. No. 4,452,901); immunoprecipitation of
labeled ligand (Brown et al. (1980) J. Biol. Chem. 255:4980-4983);
enzyme-linked immunosorbent assays (ELISA) as described, for
example, by Raines et al. (1982) J. Biol. Chem. 257:5154-5160;
immunocytochemical techniques, including the use of fluorochromes
(Brooks et al. (1980) Clin. Exp. Immunol. 39:477); and
neutralization of activity (Bowen-Pope et al. (1984) Proc. Natl.
Acad. Sci. USA 81:2396-2400). In addition to the immunoassays
described above, a number of other immunoassays are available,
including those described in U.S. Pat. Nos. 3,817,827; 3,850,752;
3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and
4,098,876.
Monoclonal antibodies that specifically bind cytokines and growth
factors (e.g., II-6, IL-12, TNF-A, IFN-.alpha., and IFN-.alpha. are
commercially available from multiple sources and can be generated
using methods known in the art (see, e.g., Kohler and Milstein,
Nature 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, A
LABORATORY MANUAL, Cold Spring Harbor Publication, New York
(1999)). Generation of monoclonal antibodies has been previously
described and can be accomplished by any means known in the art.
(Buhring et al. in Hybridoma 1991, Vol. 10, No. 1, pp. 77-78). In
some methods, the monoclonal antibody is labeled (e.g., with any
composition detectable by spectroscopic, photochemical,
biochemical, electrical, optical or chemical means) to facilitate
detection.
B. Generating siRNA
siRNA can be provided in several forms including, e.g., as one or
more isolated small-interfering RNA (siRNA) duplexes, longer
double-stranded RNA (dsRNA) or as siRNA or dsRNA transcribed from a
transcriptional cassette in a DNA plasmid. siRNA may also be
chemically synthesized. Preferably, the synthesized or transcribed
siRNA have 3' overhangs of about 1-4 nucleotides, preferably of
about 2-3 nucleotides and 5' phosphate termini. The siRNA sequences
may have overhangs (e.g., 3' or 5' overhangs as described in
(Elbashir, et al., Genes Dev. 15:188 (2001); Nykanen, et al., Cell
107:309 (2001)) or may lack overhangs (i.e., to have blunt
ends).
An RNA population can be used to provide long precursor RNAs, or
long precursor RNAs that have substantial or complete identity to a
selected target sequence can be used to make the siRNA. The RNAs
can be isolated from cells or tissue, synthesized, and/or cloned
according to methods well known to those of skill in the art. The
RNA can be a mixed population (obtained from cells or tissue,
transcribed from cDNA, subtracted, selected, etc.), or can
represent a single target sequence. RNA can be naturally occurring
(e.g., isolated from tissue or cell samples), synthesized in vitro
(e.g., using T7 or SP6 polymerase and PCR products or a cloned
CDNA); or chemically synthesized.
To form a long dsRNA, for synthetic RNAs, the complement is also
transcribed in vitro and hybridized to form a dsRNA. If a naturally
occuring RNA population is used, the RNA complements are also
provided (e.g., to form dsRNA for digestion by E. coli RNAse III or
Dicer), e.g., by transcribing cDNAs corresponding to the RNA
population, or by using RNA polymerases. The precursor RNAs are
then hybridized to form double stranded RNAs for digestion. The
dsRNAs can be directly administered to a subject or can be digested
in vitro prior to administration.
Alternatively, one or more DNA plasmids encoding one or more siRNA
templates are used to provide siRNA. siRNA can be transcribed as
sequences that automatically fold into duplexes with hairpin loops
from DNA templates in plasmids having RNA polymerase III
transcriptional units, for example, based on the naturally
occurring transcription units for small nuclear RNA U6 or human
RNase P RNA H1 (see, Brummelkamp, et al., Science 296:550 (2002);
Donze, et al., Nucleic Acids Res. 30:e46 (2002); Paddison, et al.,
Genes Dev. 16:948 (2002); Yu, et al., Proc. Natl. Acad. Sci.
99:6047 (2002); Lee, et al., Nat. Biotech. 20:500 (2002);
Miyagishi, et al., Nat. Biotech. 20:497 (2002); Paul, et al., Nat.
Biotech. 20:505 (2002); and Sui, et al., Proc. Natl. Acad. Sci.
99:5515 (2002)). Typically, a transcriptional unit or cassette will
contain an RNA transcript promoter sequence, such as an H1-RNA or a
U6 promoter, operably linked to a template for transcription of a
desired siRNA sequence and a termination sequence, comprised of 2-3
uridine residues and a polythymidine (T5) sequence (polyadenylation
signal) (Brummelkamp, Science, supra). The selected promoter can
provide for constitutive or inducible transcription. Compositions
and methods for DNA-directed transcription of RNA interference
molecules is described in detail in U.S. Pat. No. 6,573,099. The
transcriptional unit is incorporated into a plasmid or DNA vector
from which the interfering RNA is transcribed. Plasmids suitable
for in vivo delivery of genetic material for therapeutic purposes
are described in detail in U.S. Pat. Nos. 5,962,428 and 5,910,488.
The selected plasmid can provide for transient or stable delivery
of a target cell. It will be apparent to those of skill in the art
that plasmids originally designed to express desired gene sequences
can be modified to contain a transcriptional unit cassette for
transcription of siRNA.
Methods for isolating RNA, synthesizing RNA, hybridizing nucleic
acids, making and screening cDNA libraries, and performing PCR are
well known in the art (see, e.g., Gubler & Hoffman, Gene
25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra),
as are PCR methods (see U.S. Pat. No. 4,683,195 and 4,683,202; PCR
Protocols: A Guide to Methods and Applications (Innis et al., eds,
1990)). Expression libraries are also well known to those of skill
in the art. Additional basic texts disclosing the general methods
of use in this invention include Sambrook et al., Molecular
Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene
Transfer and Expression: A Laboratory Manual (1990); and Current
Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
1. Target Genes
The siRNA described herein can be used to downregulate or silence
the translation (i.e., expression) of a gene of interest. Genes of
interest include, but are not limited to, genes associated with
viral infection and survival, genes associated with metabolic
diseases and disorders (e.g., liver diseases and disorders), genes
associated with tumorigenesis and cell transformation, angiogenic
genes, immunomodulator genes, such as those associated with
inflammatory and autoimmune responses, ligand receptor genes, and
genes associated with neurodegenerative disorders.
Genes associated with viral infection and survival include those
expressed by a virus in order to bind, enter and replicate in a
cell. Of particular interest are viral sequences associated with
chronic viral diseases. Viral sequences of particular interest
include sequences of Hepatitis viruses (Hamasaki, et al., FEBS
Lett. 543:51 (2003); Yokota, et al., EMBO Rep. 4:602 (2003);
Schlomai, et al., Hepatology 37:764 (2003); Wilson, et al., Proc.
Natl. Acad. Sci. 100:2783 (2003); Kapadia, et al., Proc. Natl.
Acad. Sci. 100:2014 (2003); and FIELDS VIROLOGY (Knipe et al. eds.
2001)), Human Immunodeficiency Virus (HIV) (Banerjea, et al., Mol.
Ther. 8:62 (2003); Song, et al., J. Virol. 77:7174 (2003);
Stephenson JAMA 289:1494 (2003); Qin, et al., Proc. Natl. Acad.
Sci. 100:183 (2003)), Herpes viruses (Jia, et al., J. Virol.
77:3301 (2003)), and Human Papilloma Viruses (HPV) (Hall, et al.,
J. Virol. 77:6066 (2003); Jiang, et al., Oncogene 21:6041 (2002)).
Examplary hepatitis viral nucleic acid sequences that can be
silenced include, but are not limited to: nucleic acid sequences
involved in transcription and translation (e.g., En1, En2, X, P),
nucleic acid sequences encoding structural proteins (e.g., core
proteins including C and C-related proteins; capsid and envelope
proteins including S, M, and/or L proteins, or fragments thereof)
(see, e.g., FIELDS VIROLOGY, 2001, supra). Exemplary Hepatits C
nucleic acid sequences that can be silenced include, but are not
limited to: serine proteases (e.g., NS3/NS4), helicases (e.g. NS3),
polymerases (e.g., NS5B), and envelope proteins (e.g., E1, E2, and
p7). Hepatitis A nucleic acid sequences are set forth in e.g.,
Genbank Accession No. NC.sub.--001489; Hepatitis B nucleic acid
sequences are set forth in, e.g., Genbank Accession No.
NC.sub.--003977; Hepatitis C nucleic acid sequences are set forth
in, e.g., Genbank Accession No. NC.sub.--004102; Hepatitis D
nucleic acid sequence are set forth in, e.g., Genbank Accession No.
NC.sub.--001653; Hepatitis E nucleic acid sequences are set forth
in e.g., Genbank Accession No. NC.sub.--001434; and Hepatitis G
nucleic acid sequences are set forth in e.g., Genbank Accession No.
NC.sub.--001710. Silencing of sequences that encode genes
associated with viral infection and survival can conveniently be
used in combination with the administration of conventional agents
used to treat the viral condition.
Genes associated with metabolic diseases and disorders (e.g.,
disorders in which the liver is the target and liver diseases and
disorders) include, for example genes expressed in, for example,
dyslipidemia (e.g., liver X receptors (e.g., LXR.alpha. and
LXR.beta. Genback Accession No. NM.sub.--007121), famesoid X
receptors (FXR) (Genbank Accession No. NM.sub.--005123),
sterol-regulatory element binding protein (SREBP), Site-1 protease
(SIP), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG
coenzyme-A reductase), Apolipoprotein (ApoB), and Apolipoprotein
(ApoE)) and diabetes (e.g., Glucose 6-phosphatase) (see, e.g.,
Forman et al., Cell 81:687 (1995); Seol et al., Mol. Endocrinol.
9:72 (1995), Zavacki et al., PNAS USA 94:7909 (1997); Sakai, et
al., Cell 85:1037-1046 (1996); Duncan, et al., J. Biol. Chem.
272:12778-12785 (1997); , Willy, et al., Genes Dev. 9(9):1033-45
(1995); Lehmann, et al., J. Biol. Chem. 272(6):3137-3140 (1997);
Janowski, et al., Nature 383:728-731 (1996); Peet, et al., Cell
93:693-704 (1998)). One of skill in the art will appreciate that
genes associated with metabolic diseases and disorders (e.g.,
diseases and disorders in which the liver is a target and liver
diseases and disorders) include genes that are expressed in the
liver itself as well as and genes expressed in other organs and
tissues. Silencing of sequences that encode genes associated with
metabolic diseases and disorders can conveniently be used in
combination with the administration of conventional agents used to
treat the disease or disorder.
Examples of gene sequences associated with tumorigenesis and cell
transformation include translocation sequences such as MLL fusion
genes, BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002); Scherr, et
al., Blood 101:1566), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR,
BCL-2, AML1-ETO and AML1-MTG8 (Heidenreich, et al., Blood 101:3157
(2003)); overexpressed sequences such as multidrug resistance genes
(Nieth, et al., FEBS Lett. 545:144 (2003); Wu, et al, Cancer Res.
63:1515 (2003)), cyclins (Li, et al., Cancer Res. 63:3593 (2003);
Zou, et al., Genes Dev. 16:2923 (2002)), beta-Catenin (Verma, et
al., Clin Cancer Res. 9:1291 (2003)), telomerase genes (Kosciolek,
et al., Mol Cancer Ther. 2:209 (2003)), c-MYC, N-MYC, BCL-2, ERBB1
and ERBB2 (Nagy, et al. Exp. Cell Res. 285:39 (2003)); and mutated
sequences such as RAS (reviewed in Tuschl and Borkhardt, Mol.
Interventions, 2:158 (2002)). Silencing of sequences that encode
DNA repair enzymes find use in combination with the administration
of chemotherapeutic agents (Collis, et al., Cancer Res. 63:1550
(2003)). Genes encoding proteins associated with tumor migration
are also target sequences of interest, for example, integrins,
selectins and metalloproteinases. The foregoing examples are not
exclusive. Any whole or partial gene sequence that facilitates or
promotes tumorigenesis or cell transformation, tumor growth or
tumor migration can be included as a template sequence
Angiogenic genes are able to promote the formation of new vessels.
Of particular interest is Vascular Endothelial Growth Factor (VEGF)
(Reich, et al., Mol. Vis. 9:210 (2003)) or VEGFr. siRNA sequences
that target VEGFr are set forth in, e.g., GB 2396864; U.S. Patent
Publication No. 20040142895; and CA2456444.
Immunomodulator genes are genes that modulate one or more immune
responses. Examples of immunomodulator genes include cytokines such
as growth factors (e.g., TGF-.alpha., TGF-.beta., EGF, FGF, IGF,
NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4,
IL-12 (Hill, et al., J. Immunol. 171:691 (2003)), IL-15, IL-18,
IL-20, etc.), interferons (e.g., IFN-.alpha., IFN-.beta.,
IFN-.gamma., etc.) and TNF. Fas and Fas Ligand genes are also
immunomodulator target sequences of interest (Song, et al., Nat.
Med. 9:347 (2003)). Genes encoding secondary signaling molecules in
hematopoietic and lymphoid cells are also included in the present
invention, for example, Tec family kinases, such as Bruton's
tyrosine kinase (Btk) (Heinonen, et al., FEBS Lett. 527:274
(2002)).
Cell receptor ligands include ligands that are able to bind to cell
surface receptors (e.g., insulin receptor, EPO receptor, G-protein
coupled receptors, receptors with tyrosine kinase activity,
cytokine receptors, growth factor receptors, etc.), to modulate
(e.g., inhibit, activate, etc.) the physiological pathway that the
receptor is involved in (e.g., glucose level modulation, blood cell
development, mitogenesis, etc.). Examples of cell receptor ligands
include cytokines, growth factors, interleukins, interferons,
erythropoietin (EPO), insulin, glucagon, G-protein coupled receptor
ligands, etc.). Templates coding for an expansion of trinucleotide
repeats (e.g., CAG repeats), find use in silencing pathogenic
sequences in neurodegenerative disorders caused by the expansion of
trinucleotide repeats, such as spinobulbular muscular atrophy and
Huntington's Disease (Caplen, et al., Hum. Mol. Genet. 11:175
(2002)).
IV. SPLP Containing siRNA
In one embodiment, the present invention provides stabilized
nucleic acid-lipid particles (SPLPs or SNALPS) and other
lipid-based carrier systems containing the siRNA described herein.
As used herein, the term "SNALP" refers to a stable nucleic
acid-lipid particle, including SPLP. A SNALP represents a vesicle
of lipids coating a reduced aqueous interior comprising a nucleic
acid (e.g., ssDNA, dsDNA, ssRNA, dsRNA, siRNA, or a plasmid,
including plasmids from which an interfering RNA is transcribed).
As used herein, the term "SPLP" refers to a nucleic acid-lipid
particle comprising a nucleic acid (e.g., a plasmid) encapsulated
within a lipid vesicle. SNALPs and SPLPs typically contain a
cationic lipid, a non-cationic lipid, and a lipid that prevents
aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs
and SPLPs have systemic application as they exhibit extended
circulation lifetimes following intravenous (i.v.) injection,
accumulate at distal sites (e.g., sites physically separated from
the administration site and can mediate expression of the
transfected gene at these distal sites. SPLPs include "pSPLP" which
comprise an encapsulated condensing agent-nucleic acid complex as
set forth in WO 00/03683.
The nucleic acid-lipid particles typically comprise cationic lipid
and nucleic acids. The nucleic acid-lipid particles also preferably
comprise non-cationic lipid and a bilayer stabilizing component or,
more preferably, a conjugated lipid that inhibits aggregation of
the nucleic acid-lipid particles. The nucleic acid-lipid particles
of the present invention have a mean diameter of 50 nm to about 150
nm, more typically about 100 nm to about 130 nm, most typically
about 110 nm to about 115 nm, and are substantially nontoxic. In
addition, the nucleic acids when present in the nucleic acid-lipid
particles of the present invention are resistant in aqueous
solution to degradation with a nuclease. Such nucleic acid-lipid
particles are disclosed in great detail in U.S. Pat. Nos.
5,705,385; 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432;
and WO 96/40964.
A. Cationic Lipids
Various suitable cationic lipids may be used in the present
invention, either alone or in combination with one or more other
cationic lipid species or neutral lipid species.
Cationic lipids which are useful in the present invention can be
any of a number of lipid species which carry a net positive charge
at physiological pH, for example: DODAC, DOTMA, DDAB, DOTAP, DOSPA,
DOGS, DC-Chol and DMRIE, or combinations thereof. A number of these
lipids and related analogs, which are also useful in the present
invention, have been described in U.S. Pat. Nos. 5,208,036,
5,264,618, 5,279,833, 5,283,185, 5,753,613 and 5,785,992.
Additionally, a number of commercial preparations of cationic
lipids are available and can be used in the present invention.
These include, for example, LIPOFECTIN.RTM. (commercially available
cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand
Island, N.Y., USA); LIPOFECTAMINE.RTM. (commercially available
cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); and
TRANSFECTAM.RTM. (commercially available cationic liposomes
comprising DOGS from Promega Corp., Madison, Wis., USA). In
addition, cationic lipids of Formula I and Formula II and having
the following structures and as described in U.S. Patent
Application No. 60/578,075, filed Jun. 7, 2004:
##STR00004## and mixtures thereof can be used in the present
invention. R.sup.1 and R.sup.2 are independently selected and are
C.sub.1-C.sub.3 alkyls. R.sup.3 and R.sup.4 are independently
selected and are alkyl groups having from about 10 to about 20
carbon atoms; at least one of R.sup.3 and R.sup.4 comprises at
least two sites of unsaturation. In one embodiment, R.sup.3 and
R.sup.4 are both the same, i.e., R.sup.3 and R.sup.4 are both
linoleyl (C18), etc. In another embodiment, R.sup.3 and R.sup.4 are
different, i.e., R.sup.3is myristyl (C14) and R.sup.4 is linoleyl
(C18). In a preferred embodiment, the cationic lipids of the
present invention are symmetrical, i.e., R.sup.3 and R.sup.4 are
both the same. In another preferred embodiment, both R.sup.3 and
R.sup.4 comprise at least two sites of unsaturation. In some
embodiments, R.sup.3 and R.sup.4 are independently selected from
dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and
icosadienyl. In a preferred embodiment, R.sup.3 and R.sup.4 are
both linoleyl. In some embodiments, R.sup.3 and R.sup.4 comprise at
least three sites of unsaturation and are independently selected
from, e.g., dodecatrienyl, tetradectrienyl, hexadecatrienyl,
linolenyl, and icosatrienyl.
The cationic lipid typically comprises from about 2% to about 60%
of the total lipid present in the particle, preferably from about
5% to about 45% of the total lipid present in the particle. In
certain preferred embodiments, the cationic lipid comprises from
about 5% to about 15% of the total lipid present in the particle.
In other preferred embodiments, the cationic lipid comprises from
about 40% to about 50% of the total lipid present in the particle.
Depending on the intended use of the nucleic acid-lipid particles,
the proportions of the components are varied and the delivery
efficiency of a particular formulation can be measured using an
endosomal release parameter (ERP) assay. For example, for systemic
delivery, the cationic lipid may comprise from about 5% to about
15% of the total lipid present in said particle and for local or
regional delivery, the cationic lipid comprises from about 40% to
about 50% of the total lipid present in said particle.
B. Non-cationic Lipids
The non-cationic lipids used in the present invention can be any of
a variety of neutral uncharged, zwitterionic or anionic lipids
capable of producing a stable complex. They are preferably neutral,
although they can alternatively be positively or negatively
charged. Examples of non-cationic lipids useful in the present
invention include: phospholipid-related materials, such as
lecithin, phosphatidylethanolamine, lysolecithin,
lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cephalin, cardiolipin,
phosphatidic acid, cerebrosides, dicetylphosphate,
phospholipid-related materials, such as lecithin,
phosphatidylethanolamine, lysolecithin,
lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cephalin, cardiolipin,
phosphatidic acid, cerebrosides, dicetylphosphate,
distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE). Non-cationic
lipids or sterols such as cholesterol may be present. Additional
nonphosphorous containing lipids are, e.g., stearylamine,
dodecylamine, hexadecylamine, acetyl palmitate,
glycerolricinoleate, hexadecyl stereate, isopropyl myristate,
amphoteric acrylic polymers, triethanolamine-lauryl sulfate,
alkyl-aryl sulfate polyethyloxylated fatty acid amides,
dioctadecyldimethyl ammonium bromide and the like,
diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, and cerebrosides. Other lipids
such as lysophosphatidylcholine and lysophosphatidylethanolamine
may be present. Non-cationic lipids also include polyethylene
glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene
glycol conjugated to phospholipids or to ceramides (referred to as
PEG-Cer), as described in U.S. Pat. No. 5,820,873.
In preferred embodiments, the non-cationic lipids are
diacylphosphatidylcholine (e.g., distearoylphosphatidylcholine,
dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine and
dilinoleoylphosphatidylcholine), diacylphosphatidylethanolamine
(e.g., dioleoylphosphatidylethanolamine and
palmitoyloleoylphosphatidylethanolamine), ceramide or
sphingomyelin. The acyl groups in these lipids are preferably acyl
groups derived from fatty acids having C.sub.10-C.sub.24 carbon
chains. More preferably the acyl groups are lauroyl, myristoyl,
palmitoyl, stearoyl or oleoyl. In particularly preferred
embodiments, the non-cationic lipid will include one or more of
cholesterol, 1,2-sn-dioleoylphosphatidylethanolamine, or egg
sphingomyelin (ESM).
The non-cationic lipid typically comprises from about 5% to about
90% of the total lipid present in the particle, preferably from
about 20% to about 85% of the total lipid present in tje particle.
The nucleic acid-lipid particles of the present invention may
further comprise a sterol (e.g., cholesterol). If present, the
cholesterol typically comprises from about 10% to about 60% of the
total lipid present in the particle, preferably the cholesterol
comprises from about 20% to about 45% of the total lipid present in
the particle.
C. Bilayer Stabilizing Components
In one embodiment, the nucleic acid-lipid particle (e.g., SPLP, or
SNLAP) further comprises a bilayer stabilizing component (BSC)
(i.e., a conjugated lipid that prevents aggregation of particles).
Bilayer stabilizing Suitable BSCs include, but are not limited to,
polyamide oligomers, peptides, proteins, detergents,
lipid-derivatives, PEG-lipids, such as PEG coupled to
dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol
(PEG-DAG), PEG coupled to phosphatidylethanolamine (PE) (PEG-PE),
or PEG conjugated to ceramides (PEG-Cer), or a mixture thereof
(see, U.S. Pat. No. 5,885,613). In one embodiment, the bilayer
stabilizing component is a PEG-lipid, or an ATTA-lipid. In one
preferred embodiment, the BSC is a conjugated lipid that inhibits
aggregation of the SNALPs. Suitable conjugated lipids include, but
are not limited to PEG-lipid conjugates, ATTA-lipid conjugates,
cationic-polymer-lipid conjugates (CPLs) or mixtures thereof. In
one preferred embodiment, the SNALPs comprise either a PEG-lipid
conjugate or an ATTA-lipid conjugate together with a CPL.
In one embodiment, the bilayer stabilizing component comprises a
diacylglycerol-polyethyleneglycol conjugate, i.e., a DAG-PEG
conjugate or a PEG-DAG conjugate. In a preferred embodiment, the
DAG-PEG conjugate is a dilaurylglycerol (C.sub.12)-PEG conjugate,
dimyristylglycerol (C.sub.14)-PEG conjugate (DMG), a
dipalmitoylglycerol (C.sub.16)-PEG conjugate or a distearylglycerol
(C.sub.18)-PEG conjugate (DSG). Those of skill in the art will
readily appreciate that other diacylglycerols can be used in the
DAG-PEG conjugates of the present invention. Suitable DAG-PEG
conjugates for use in the present invention, and methods of making
and using them, are disclosed in U.S. Patent Publication No.
2003/0077829, and PCT Patent Application No. CA 02/00669.
In another embodiment, the bilayer stabilizing component comprises
a dialkyloxypropyl conjugate, i.e., a PEG-DAA conjugate as
described in, e.g., U.S. Patent Application Nos. 60/503,329, filed
Sep. 15, 2003 and Ser. No. 10/942,379, filed Sep. 15, 2004. In one
preferred embodiment, the PEG-DAA conjugate has the following
formula:
##STR00005##
In Formula III above, "R.sup.1 and R.sup.2" are independently
selected and are saturated or unsaturated alkyl groups having from
about 10 to about 20 carbon atoms; PEG is a polyethyleneglycol; and
L is a non-ester-containing linker moiety as described above.
Suitable alkyl groups include, but are not limited to, lauryl
(C12), myristyl (C14), palmityl (C16), stearyl (C18) and icosyl
(C20). In a preferred embodiment; R.sup.1 and R.sup.2 are the same,
i.e., they are both myristyl (C14) or both palmityl (C16) or both
stearyl (C18). In a preferred embodiment, the alkyl groups are
saturated.
In Formula III above, "PEG" is a polyethylene glycol having an
average molecular weight ranging of about 550 daltons to about
10,000 daltons, more preferably of about 750 daltons to about 5,000
daltons, more preferably of about 1,000 daltons to about 5,000
daltons, more preferably of about 1,500 daltons to about 3,000
daltons and, even more preferably, of about 2,000 daltons, or about
750 daltons. The PEG can be optionally substituted with alkyl,
alkoxy, acyl or aryl. In a preferred embodiment, the terminal
hydroxyl group is substituted with a methoxy or methyl group.
In Formula III, above, "L" is a non-ester containing linker moiety
or an ester containing linker moiety. In a preferred embodiment, L
is a non-ester containing linker moiety. Suitable non-ester
containing linkers include, but are not limited to, an amido linker
moiety, an amino linker moiety, a carbonyl linker moiety, a
carbamate linker moiety, a urea linker moiety, an ether linker
moiety, a disulphide linker moiety, a succinamidyl linker moiety
and combinations thereof. In a preferred embodiment, the non-ester
containing linker moiety is a carbamate linker moiety (i.e., a
PEG-C-DAA conjugate). In another preferred embodiment, the
non-ester containing linker moiety is an amido linker moiety (i.e.,
a PEG-A-DAA conjugate). In a preferred embodiment, the non-ester
containing linker moiety is a succinamidyl linker moiety (i.e., a
PEG-S-DAA conjugate).
Phosphatidylethanolamines having a variety of acyl chain groups of
varying chain lengths and degrees of saturation can be conjugated
to polyethyleneglycol to form the bilayer stabilizing component.
Such phosphatidylethanolamines are commercially available, or can
be isolated or synthesized using conventional techniques known to
those of skilled in the art. Phosphatidylethanolamines containing
saturated or unsaturated fatty acids with carbon chain lengths in
the range of C.sub.10 to C.sub.20 are preferred.
Phosphatidylethanolamines with mono- or diunsaturated fatty acids
and mixtures of saturated and unsaturated fatty acids can also be
used. Suitable phosphatidylethanolamines include, but are not
limited to, the following: dimyristoylphosphatidylethanolamine
(DMPE), dipalmitoylphosphatidylethanolamine (DPPE),
dioleoylphosphatidylethanolamine (DOPE) and
distearoylphosphatidylethanolamine (DSPE).
As with the phosphatidylethanolamines, ceramides having a variety
of acyl chain groups of varying chain lengths and degrees of
saturation can be coupled to polyethyleneglycol to form the bilayer
stabilizing component. It will be apparent to those of skill in the
art that in contrast to the phosphatidylethanolamines, ceramides
have only one acyl group which can be readily varied in terms of
its chain length and degree of saturation. Ceramides suitable for
use in accordance with the present invention are commercially
available. In addition, ceramides can be isolated, for example,
from egg or brain using well-known isolation techniques or,
alternatively, they can be synthesized using the methods and
techniques disclosed in U.S. Pat. No. 5,820,873. Using the
synthetic routes set forth in the foregoing application, ceramides
having saturated or unsaturated fatty acids with carbon chain
lengths in the range of C.sub.2 to C.sub.31 can be prepared.
Cationic polymer lipids (CPLs) useful in the present invention have
the following architectural features: (1) a lipid anchor, such as a
hydrophobic lipid, for incorporating the CPLs into the lipid
bilayer; (2) a hydrophilic spacer, such as a polyethylene glycol,
for linking the lipid anchor to a cationic head group; and (3) a
polycationic moiety, such as a naturally occurring amino acid, to
produce a protonizable cationic head group. Suitable CPLs for use
in the present invention, and methods of making and using nucleic
acid-lipid particles comprising the CPLs, are disclosed, e.g., in
U.S. Pat. No. 6,852,334; U.S. Patent Publication No. 20020072121;
and WO 00/62813).
Briefly, CPL's suitable for use in the present invention include
compounds of Formula IV: A-W-Y (IV) wherein A, W and Y are as
follows.
With reference to Formula IV, "A" is a lipid moiety such as an
amphipathic lipid, a neutral lipid or a hydrophobic lipid that acts
as a lipid anchor. Suitable lipid examples include vesicle-forming
lipids or vesicle adopting lipids and include, but are not limited
to, diacylglycerolyls, dialkylglycerolyls, N-N-dialkylaminos,
1,2-diacyloxy-3-aminopropanes and 1,2-dialkyl-3-aminopropanes.
"W" is a polymer or an oligomer, such as a hydrophilic polymer or
oligomer. Preferably, the hydrophilic polymer is a biocompatable
polymer that is nonimmunogenic or possesses low inherent
immunogenicity. Alternatively, the hydrophilic polymer can be
weakly antigenic if used with appropriate adjuvants. Suitable
nonimmunogenic polymers include, but are not limited to, PEG,
polyamides, polylactic acid, polyglycolic acid, polylactic
acid/polyglycolic acid copolymers and combinations thereof. In a
preferred embodiment, the polymer has a molecular weight of about
250 to about 7000 daltons.
"Y" is a polycationic moiety. The term polycationic moiety refers
to a compound, derivative, or functional group having a positive
charge, preferably at least 2 positive charges at a selected pH,
preferably physiological pH. Suitable polycationic moieties include
basic amino acids and their derivatives such as arginine,
asparagine, glutamine, lysine and histidine; spermine; spermidine;
cationic dendrimers; polyamines; polyamine sugars; and amino
polysaccharides. The polycationic moieties can be linear, such as
linear tetralysine, branched or dendrimeric in structure.
Polycationic moieties have between about 2 to about 15 positive
charges, preferably between about 2 to about 12 positive charges,
and more preferably between about 2 to about 8 positive charges at
selected pH values. The selection of which polycationic moiety to
employ may be determined by the type of liposome application which
is desired.
The charges on the polycationic moieties can be either distributed
around the entire liposome moiety or, alternatively, they can be a
discrete concentration of charge density in one particular area of
the liposome moiety e.g., a charge spike. If the charge density is
distributed on the liposome, the charge density can be equally
distributed or unequally distributed. All variations of charge
distribution of the polycationic moiety are encompassed by the
present invention.
The lipid "A," and the nonimmunogenic polymer "W," can be attached
by various methods and preferably, by covalent attachment. Methods
known to those of skill in the art can be used for the covalent
attachment of "A" and "W." Suitable linkages include, but are not
limited to, amide, amine, carboxyl, carbonate, carbamate, ester and
hydrazone linkages. It will be apparent to those skilled in the art
that "A" and "'W" must have complementary functional groups to
effectuate the linkage. The reaction of these two groups, one on
the lipid and the other on the polymer, will provide the desired
linkage. For example, when the lipid is a diacylglycerol and the
terminal hydroxyl is activated, for instance with NHS and DCC, to
form an active ester, and is then reacted with a polymer which
contains an amino group, such as with a polyamide (see, U.S. Pat.
Nos. 6,320,017 and 6,586,559), an amide bond will form between the
two groups.
In certain instances, the polycationic moiety can have a ligand
attached, such as a targeting ligand or a chelating moiety for
complexing calcium. Preferably, after the ligand is attached, the
cationic moiety maintains a positive charge. In certain instances,
the ligand that is attached has a positive charge. Suitable ligands
include, but are not limited to, a compound or device with a
reactive functional group and include lipids, amphipathic lipids,
carrier compounds, bioaffinity compounds, biomaterials,
biopolymers, biomedical devices, analytically detectable compounds,
therapeutically active compounds, enzymes, peptides, proteins,
antibodies, immune stimulators, radiolabels, fluorogens, biotin,
drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes,
micelles, immunoglobulins, functional groups, other targeting
moieties, or toxins.
Typically, the bilayer stabilizing component is present ranging
from about 0.5% to about 50% of the total lipid present in the
particle. In a preferred embodiment, the bilayer stabilizing
component is present from about 0.5% to about 25% of the total
lipid in the particle. In other preferred embodiments, the bilayer
stabilizing component is present from about 1% to about 20%, or
about 3% to about 15% or about 4% to about 10% of the total lipid
in the particle. One of ordinary skill in the art will appreciate
that the concentration of the bilayer stabilizing component can be
varied depending on the bilayer stabilizing component employed and
the rate at which the liposome is to become fusogenic.
By controlling the composition and concentration of the bilayer
stabilizing component, one can control the rate at which the
bilayer stabilizing component exchanges out of the liposome and, in
turn, the rate at which the liposome becomes fusogenic. For
instance, when a polyethyleneglycol-phosphatidylethanolamine
conjugate or a polyethyleneglycol-ceramide conjugate is used as the
bilayer stabilizing component, the rate at which the liposome
becomes fusogenic can be varied, for example, by varying the
concentration of the bilayer stabilizing component, by varying the
molecular weight of the polyethyleneglycol, or by varying the chain
length and degree of saturation of the acyl chain groups on the
phosphatidylethanolamine or the ceramide. In addition, other
variables including, for example, pH, temperature, ionic strength,
etc. can be used to vary and/or control the rate at which the
liposome becomes fusogenic. Other methods which can be used to
control the rate at which the liposome becomes fusogenic will
become apparent to those of skill in the art upon reading this
disclosure.
V. Preparation of Nucleic Acid-Lipid Particles
The present invention provides a method of preparing serum-stable
nucleic acid-lipid particles in which the plasmid or other nucleic
acid is encapsulated in a lipid bilayer and is protected from
degradation. The particles made by the methods of this invention
typically have a size of about 50 nm to about 150 nm, more
typically about 100 nm to about 130 nm, most typically about 110 nm
to about 115 nm. The particles can be formed by any method known in
the art including, but not limited to: a continuous mixing method,
a detergent dialysis method, or a modification of a reverse-phase
method which utilizes organic solvents to provide a single phase
during mixing of the components.
In a particularly preferred embodiment, the present invention
provides for nucleic acid-lipid particles produced via a continuous
mixing method, e.g., process that includes providing an aqueous
solution comprising a nucleic acid such as an siRNA or a plasmid,
in a first reservoir, and providing an organic lipid solution in a
second reservoir, and mixing the aqueous solution with the organic
lipid solution such that the organic lipid solution mixes with the
aqueous solution so as to substantially instantaneously produce a
liposome encapsulating the nucleic acid (e.g., siRNA). This process
and the apparatus for carrying this process is described in detail
in U.S. Patent Publication No. 20040142025.
The action of continuously introducing lipid and buffer solutions
into a mixing environment, such as in a mixing chamber, causes a
continuous dilution of the lipid solution with the buffer solution,
thereby producing a liposome substantially instantaneously upon
mixing. As used herein, the phrase "continuously diluting a lipid
solution with a buffer solution" (and variations) generally means
that the lipid solution is diluted sufficiently rapidly in a
hydration process with sufficient force to effectuate vesicle
generation. By mixing the aqueous solution comprising a nucleic
acid with the organic lipid solution, the organic lipid solution
undergoes a continuous stepwise dilution in the presence of the
buffer solution (i.e., aqueous solution) to produce a nucleic
acid-lipid particle.
In some embodiments, the particles are formed using detergent
dialysis. Without intending to be bound by any particular mechanism
of formation, a plasmid or other nucleic acid (e.g., siRNA) is
contacted with a detergent solution of cationic lipids to form a
coated nucleic acid complex. These coated nucleic acids can
aggregate and precipitate. However, the presence of a detergent
reduces this aggregation and allows the coated nucleic acids to
react with excess lipids (typically, non-cationic lipids) to form
particles in which the plasmid or other nucleic acid is
encapsulated in a lipid bilayer. Thus, the present invention
provides a method for the preparation of serum-stable nucleic
acid-lipid particles, comprising: (a) combining a nucleic acid with
cationic lipids in a detergent solution to form a coated nucleic
acid-lipid complex; (b) contacting non-cationic lipids with the
coated nucleic acid-lipid complex to form a detergent solution
comprising a nucleic acid-lipid complex and non-cationic lipids;
and (c) dialyzing the detergent solution of step (b) to provide a
solution of serum-stable nucleic acid-lipid particles, wherein the
nucleic acid is encapsulated in a lipid bilayer and the particles
are serum-stable and have a size of from about 50 to about 150
nm.
An initial solution of coated nucleic acid-lipid complexes is
formed by combining the nucleic acid with the cationic lipids in a
detergent solution.
In these embodiments, the detergent solution is preferably an
aqueous solution of a neutral detergent having a critical micelle
concentration of 15-300 mM, more preferably 20-50 mM. Examples of
suitable detergents include, for example,
N,N'-((octanoylimino)-bis-(trimethylene))-bis-(D-gluconamide)
(BIGCHAP); BRIJ 35; Deoxy-BIGCHAP; dodecylpoly(ethylene glycol)
ether; Tween 20; Tween 40; Tween 60; Tween 80; Tween 85; Mega 8;
Mega 9; Zwittergent.RTM. 3-08; Zwittergent.RTM. 3-10; Triton X-405;
hexyl-, heptyl-,octyl- and nonyl-.beta.-D-glucopyranoside; and
heptylthioglucopyranoside; with octyl .beta.-D-glucopyranoside and
Tween-20 being the most preferred. The concentration of detergent
in the detergent solution is typically about 100 mM to about 2 M,
preferably from about 200 mM to about 1.5 M.
The cationic lipids and nucleic acids will typically be combined to
produce a charge ratio (+/-) of about 1:1 to about 20:1, preferably
in a ratio of about 1:1 to about 12:1, and more preferably in a
ratio of about 2:1 to about 6:1. Additionally, the overall
concentration of nucleic acid in solution will typically be from
about 25 .mu.g/mL to about 1 mg/mL, preferably from about 25
.mu.g/mL to about 200 .mu.g/mL, and more preferably from about 50
.mu.g/mL to about 100 .mu.g/mL. The combination of nucleic acids
and cationic lipids in detergent solution is kept, typically at
room temperature, for a period of time which is sufficient for the
coated complexes to form. Alternatively, the nucleic acids and
cationic lipids can be combined in the detergent solution and
warmed to temperatures of up to about 37.degree. C. For nucleic
acids which are particularly sensitive to temperature, the coated
complexes can be formed at lower temperatures, typically down to
about 4.degree. C.
In a preferred embodiment, the nucleic acid to lipid ratios
(mass/mass ratios) in a formed nucleic acid-lipid particle will
range from about 0.01 to about 0.08. The ratio of the starting
materials also falls within this range because the purification
step typically removes the unencapsulated nucleic acid as well as
the empty liposomes. In another preferred embodiment, the nucleic
acid-lipid particle preparation uses about 400 .mu.g nucleic acid
per 10 mg total lipid or a nucleic acid to lipid ratio of about
0.01 to about 0.08 and, more preferably, about 0.04, which
corresponds to 1.25 mg of total lipid per 50 .mu.g of nucleic
acid.
The detergent solution of the coated nucleic acid-lipid complexes
is then contacted with non-cationic lipids to provide a detergent
solution of nucleic acid-lipid complexes and non-cationic lipids.
The non-cationic lipids which are useful in this step include,
diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, cardiolipin, and cerebrosides.
In preferred embodiments, the non-cationic lipids are
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide
or sphingomyelin. The acyl groups in these lipids are preferably
acyl groups derived from fatty acids having C.sub.10-C.sub.24
carbon chains. More preferably the acyl groups are lauroyl,
myristoyl, palmitoyl, stearoyl or oleoyl. In particularly preferred
embodiments, the non-cationic lipid will be
1,2-sn-dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoyl
phosphatidylcholine (POPC), egg phosphatidylcholine (EPC),
distearoylphosphatidylcholine (DSPC), cholesterol, or a mixture
thereof. In the most preferred embodiments, the nucleic acid-lipid
particles will be fusogenic particles with enhanced properties in
vivo and the non-cationic lipid will be DSPC or DOPE. In addition,
the nucleic acid-lipid particles of the present invention may
further comprise cholesterol. In other preferred embodiments, the
non-cationic lipids will further comprise polyethylene glycol-based
polymers such as PEG 2000, PEG 5000 and polyethylene glycol
conjugated to a diacylglycerol, a ceramide or a phospholipid, as
described in U.S. Pat. No. 5,820,873 and U.S. Patent Publication
No. 20030077829. In further preferred embodiments, the non-cationic
lipids will further comprise polyethylene glycol-based polymers
such as PEG 2000, PEG 5000 and polyethylene glycol conjugated to a
dialkyloxypropyl.
The amount of non-cationic lipid which is used in the present
methods is typically about 2 to about 20 mg of total lipids to 50
.mu.g of nucleic acid. Preferably the amount of total lipid is from
about 5 to about 10 mg per 50 .mu.g of nucleic acid.
Following formation of the detergent solution of nucleic acid-lipid
complexes and non-cationic lipids, the detergent is removed,
preferably by dialysis. The removal of the detergent results in the
formation of a lipid-bilayer which surrounds the nucleic acid
providing serum-stable nucleic acid-lipid particles which have a
size of from about 50 nm to about 150 nm, more typically about 100
nm to about 130 nm, most typically about 110 nm to about 115 nm.
The particles thus formed do not aggregate and are optionally sized
to achieve a uniform particle size.
The serum-stable nucleic acid-lipid particles can be sized by any
of the methods available for sizing liposomes. The sizing may be
conducted in order to achieve a desired size range and relatively
narrow distribution of particle sizes.
Several techniques are available for sizing the particles to a
desired size. One sizing method, used for liposomes and equally
applicable to the present particles is described in U.S. Pat. No.
4,737,323. Sonicating a particle suspension either by bath or probe
sonication produces a progressive size reduction down to particles
of less than about 50 nm in size. Homogenization is another method
which relies on shearing energy to fragment larger particles into
smaller ones. In a typical homogenization procedure, particles are
recirculated through a standard emulsion homogenizer until selected
particle sizes, typically between about 60 and 80 nm, are observed.
In both methods, the particle size distribution can be monitored by
conventional laser-beam particle size discrimination, or QELS.
Extrusion of the particles through a small-pore polycarbonate
membrane or an asymmetric ceramic membrane is also an effective
method for reducing particle sizes to a relatively well-defined
size distribution. Typically, the suspension is cycled through the
membrane one or more times until the desired particle size
distribution is achieved. The particles may be extruded through
successively smaller-pore membranes, to achieve a gradual reduction
in size.
In another group of embodiments, the present invention provides a
method for the preparation of serum-stable nucleic acid-lipid
particles, comprising: (a) preparing a mixture comprising cationic
lipids and non-cationic lipids in an organic solvent; (b)
contacting an aqueous solution of nucleic acid with said mixture in
step (a) to provide a clear single phase; and (c) removing said
organic solvent to provide a suspension of nucleic acid-lipid
particles, wherein said nucleic acid is encapsulated in a lipid
bilayer, and said particles are stable in serum and have a size of
from about 50 to about 150 nm.
The nucleic acids (or plasmids), cationic lipids and non-cationic
lipids which are useful in this group of embodiments are as
described for the detergent dialysis methods above.
The selection of an organic solvent will typically involve
consideration of solvent polarity and the ease with which the
solvent can be removed at the later stages of particle formation.
The organic solvent, which is also used as a solubilizing agent, is
in an amount sufficient to provide a clear single phase mixture of
nucleic acid and lipids. Suitable solvents include, but are not
limited to, chloroform, dichloromethane, diethylether, cyclohexane,
cyclopentane, benzene, toluene, methanol, or other aliphatic
alcohols such as propanol, isopropanol, butanol, tert-butanol,
iso-butanol, pentanol and hexanol. Combinations of two or more
solvents may also be used in the present invention.
Contacting the nucleic acid with the organic solution of cationic
and non-cationic lipids is accomplished by mixing together a first
solution of nucleic acid, which is typically an aqueous solution,
and a second organic solution of the lipids. One of skill in the
art will understand that this mixing can take place by any number
of methods, for example by mechanical means such as by using vortex
mixers.
After the nucleic acid has been contacted with the organic solution
of lipids, the organic solvent is removed, thus forming an aqueous
suspension of serum-stable nucleic acid-lipid particles. The
methods used to remove the organic solvent will typically involve
evaporation at reduced pressures or blowing a stream of inert gas
(e.g., nitrogen or argon) across the mixture.
The serum-stable nucleic acid-lipid particles thus formed will
typically be sized from about 50 nm to about 150 nm, more typically
about 100 nm to about 130 nm, most typically about 110 nm to about
115 nm. To achieve further size reduction or homogeneity of size in
the particles, sizing can be conducted as described above.
In other embodiments, the methods will further comprise adding
nonlipid polycations which are useful to effect the delivery to
cells using the present compositions. Examples of suitable nonlipid
polycations include, but are limited to, hexadimethrine bromide
(sold under the brandname POLYBRENE.RTM., from Aldrich Chemical
Co., Milwaukee, Wis., USA) or other salts of heaxadimethrine. Other
suitable polycations include, for example, salts of
poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,
polyallylamine and polyethyleneimine.
In certain embodiments, the formation of the nucleic acid-lipid
particles can be carried out either in a mono-phase system (e.g., a
Bligh and Dyer monophase or similar mixture of aqueous and organic
solvents) or in a two-phase system with suitable mixing.
When formation of the complexes is carried out in a mono-phase
system, the cationic lipids and nucleic acids are each dissolved in
a volume of the mono-phase mixture. Combination of the two
solutions provides a single mixture in which the complexes form.
Alternatively, the complexes can form in two-phase mixtures in
which the cationic lipids bind to the nucleic acid (which is
present in the aqueous phase), and "pull" it into the organic
phase.
In another embodiment, the present invention provides a method for
the preparation of nucleic acid-lipid particles, comprising: (a)
contacting nucleic acids with a solution comprising non-cationic
lipids and a detergent to form a nucleic acid-lipid mixture; (b)
contacting cationic lipids with the nucleic acid-lipid mixture to
neutralize a portion of the negative charge of the nucleic acids
and form a charge-neutralized mixture of nucleic acids and lipids;
and (c) removing the detergent from the charge-neutralized mixture
to provide the nucleic acid-lipid particles in which the nucleic
acids are protected from degradation.
In one group of embodiments, the solution of non-cationic lipids
and detergent is an aqueous solution. Contacting the nucleic acids
with the solution of non-cationic lipids and detergent is typically
accomplished by mixing together a first solution of nucleic acids
and a second solution of the lipids and detergent. One of skill in
the art will understand that this mixing can take place by any
number of methods, for example, by mechanical means such as by
using vortex mixers. Preferably, the nucleic acid solution is also
a detergent solution. The amount of non-cationic lipid which is
used in the present method is typically determined based on the
amount of cationic lipid used, and is typically of from about 0.2
to 5 times the amount of cationic lipid, preferably from about 0.5
to about 2 times the amount of cationic lipid used.
In some embodiments, the nucleic acids are precondensed as
described in, e.g., U.S. patent application Ser. No.
09/744,103.
The nucleic acid-lipid mixture thus formed is contacted with
cationic lipids to neutralize a portion of the negative charge
which is associated with the nucleic acids (or other polyanionic
materials) present. The amount of cationic lipids used will
typically be sufficient to neutralize at least 50% of the negative
charge of the nucleic acid. Preferably, the negative charge will be
at least 70% neutralized, more preferably at least 90% neutralized.
Cationic lipids which are useful in the present invention, include,
for example, DODAC, DOTMA, DDAB, DOTAP, DC-Chol and DMRIE. These
lipids and related analogs have been described in U.S. Pat. Nos.
5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613 and
5,785,992.
Contacting the cationic lipids with the nucleic acid-lipid mixture
can be accomplished by any of a number of techniques, preferably by
mixing together a solution of the cationic lipid and a solution
containing the nucleic acid-lipid mixture. Upon mixing the two
solutions (or contacting in any other manner), a portion of the
negative charge associated with the nucleic acid is neutralized.
Nevertheless, the nucleic acid remains in an uncondensed state and
acquires hydrophilic characteristics.
After the cationic lipids have been contacted with the nucleic
acid-lipid mixture, the detergent (or combination of detergent and
organic solvent) is removed, thus forming the nucleic acid-lipid
particles. The methods used to remove the detergent will typically
involve dialysis. When organic solvents are present, removal is
typically accomplished by evaporation at reduced pressures or by
blowing a stream of inert gas (e.g., nitrogen or argon) across the
mixture.
The particles thus formed will typically be sized from about 50 nm
to several microns, more typically about 50 nm to about 150 nm,
even more typically about 100 nm to about 130 nm, most typically
about 110 nm to about 115 nm. To achieve further size reduction or
homogeneity of size in the particles, the nucleic acid-lipid
particles can be sonicated, filtered or subjected to other sizing
techniques which are used in liposomal formulations and are known
to those of skill in the art.
In other embodiments, the methods will further comprise adding
nonlipid polycations which are useful to effect the lipofection of
cells using the present compositions. Examples of suitable nonlipid
polycations include, hexadimethrine bromide (sold under the
brandname POLYBRENE.RTM., from Aldrich Chemical Co., Milwaukee,
Wis., USA) or other salts of hexadimethrine. Other suitable
polycations include, for example, salts of poly-L-ornithine,
poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine and
polyethyleneimine. Addition of these salts is preferably after the
particles have been formed.
In another aspect, the present invention provides methods for the
preparation of nucleic acid-lipid particles, comprising: (a)
contacting an amount of cationic lipids with nucleic acids in a
solution; the solution comprising from about 15-35% water and about
65-85% organic solvent and the amount of cationic lipids being
sufficient to produce a +/- charge ratio of from about 0.85 to
about 2.0, to provide a hydrophobic nucleic acid-lipid complex;
(b)contacting the hydrophobic, nucleic acid-lipid complex in
solution with non-cationic lipids, to provide a nucleic acid-lipid
mixture; and (c) removing the organic solvents from the nucleic
acid-lipid mixture to provide nucleic acid-lipid particles in which
the nucleic acids are protected from degradation.
The nucleic acids, non-cationic lipids, cationic lipids and organic
solvents which are useful in this aspect of the invention are the
same as those described for the methods above which used
detergents. In one group of embodiments, the solution of step (a)
is a mono-phase. In another group of embodiments, the solution of
step (a) is two-phase.
In preferred embodiments, the cationic lipids are DODAC, DDAB,
DOTMA, DOSPA, DMRIE, DOGS or combinations thereof. In other
preferred embodiments, the non-cationic lipids are ESM, DOPE, DOPC,
DSPC, polyethylene glycol-based polymers (e.g., PEG 2000, PEG 5000,
PEG-modified diacylglycerols, or PEG-modified dialkyloxypropyls),
distearoylphosphatidylcholine (DSPC), cholesterol, or combinations
thereof. In still other preferred embodiments, the organic solvents
are methanol, chloroform, methylene chloride, ethanol, diethyl
ether or combinations thereof.
In one embodiment, the nucleic acid is a plasmid from which an
interfering RNA is transcribed; the cationic lipid is DODAC, DDAB,
DOTMA, DOSPA, DMRIE, DOGS or combinations thereof; the non-cationic
lipid is ESM, DOPE, DAG-PEGs, distearoylphosphatidylcholine (DSPC),
cholesterol, or combinations thereof (e.g. DSPC and DAG-PEGs); and
the organic solvent is methanol, chloroform, methylene chloride,
ethanol, diethyl ether or combinations thereof.
As above, contacting the nucleic acids with the cationic lipids is
typically accomplished by mixing together a first solution of
nucleic acids and a second solution of the lipids, preferably by
mechanical means such as by using vortex mixers. The resulting
mixture contains complexes as described above. These complexes are
then converted to particles by the addition of non-cationic lipids
and the removal of the organic solvent. The addition of the
non-cationic lipids is typically accomplished by simply adding a
solution of the non-cationic lipids to the mixture containing the
complexes. A reverse addition can also be used. Subsequent removal
of organic solvents can be accomplished by methods known to those
of skill in the art and also described above.
The amount of non-cationic lipids which is used in this aspect of
the invention is typically an amount of from about 0.2 to about 15
times the amount (on a mole basis) of cationic lipids which was
used to provide the charge-neutralized nucleic acid-lipid complex.
Preferably, the amount is from about 0.5 to about 9 times the
amount of cationic lipids used.
In yet another aspect, the present invention provides nucleic
acid-lipid particles which are prepared by the methods described
above. In these embodiments, the nucleic acid-lipid particles are
either net charge neutral or carry an overall charge which provides
the particles with greater gene lipofection activity. Preferably,
the nucleic acid component of the particles is a nucleic acid which
interferes with the production of an undesired protein. In a
preferred embodiment, the nucleic acid comprises an interfering
RNA, the non-cationic lipid is egg sphingomyelin and the cationic
lipid is DODAC. In a preferred embodiment, the nucleic acid
comprises an interfering RNA, the non-cationic lipid is a mixture
of DSPC and cholesterol, and the cationic lipid is DOTMA. In other
preferred embodiments, the non-cationic lipid may further comprise
cholesterol.
A variety of general methods for making SNALP-CPLs (CPL-containing
SNALPs) are discussed herein. Two general techniques include
"post-insertion" technique, that is, insertion of a CPL into for
example, a pre-formed SNALP, and the "standard" technique, wherein
the CPL is included in the lipid mixture during for example, the
SNALP formation steps. The post-insertion technique results in
SNALPs having CPLs mainly in the external face of the SNALP bilayer
membrane, whereas standard techniques provide SNALPs having CPLs on
both internal and external faces. The method is especially useful
for vesicles made from phospholipids (which can contain
cholesterol) and also for vesicles containing PEG-lipids (such as
PEG-DAGs). Methods of making SNALP-CPL, are taught, for example, in
U.S. Pat. Nos. 5,705,385, 6,586,410, 5,981,501 6,534,484;
6,852,334; U.S. Patent Publication No. 20020072121, as well as in
WO 00/62813.
VI. Kits
The present invention also provides nucleic acid-lipid particles in
kit form. The kit may comprise a container which is
compartmentalized for holding the various elements of the nucleic
acid-lipid particles (e.g., the nucleic acids and the individual
lipid components of the particles). In some embodiments, the kit
contains the nucleic acid-lipid particles compositions of the
present inventions, preferably in dehydrated form, with
instructions for their rehydration and administration.
VII. Administration of Nucleic Acid-Lipid Particles
The serum-stable nucleic acid-lipid particles of the present
invention are useful for the introduction of nucleic acids into
cells. Accordingly, the present invention also provides methods for
introducing a nucleic acids (e.g., an interfering RNA) into a cell.
Depending on the desired effect, the immunostimulatory effects of
the siRNA can be enhanced or diminished by introducing (i.e.,
enhance) or eliminating (i.e., diminish) the 5'-GU'3' dinucleotide
motif. The methods are carried out in vitro or in vivo by first
forming the particles as described above, then contacting the
particles with the cells for a period of time sufficient for
delivery of interfering RNA to occur.
The nucleic acid-lipid particles of the present invention can be
adsorbed to almost any cell type with which they are mixed or
contacted. Once adsorbed, the particles can either be endocytosed
by a portion of the cells, exchange lipids with cell membranes, or
fuse with the cells. Transfer or incorporation of the nucleic acid
portion of the particle can take place via any one of these
pathways. In particular, when fusion takes place, the particle
membrane is integrated into the cell membrane and the contents of
the particle combine with the intracellular fluid.
A. In vitro Delivery
For in vitro applications, the delivery of nucleic acids can be to
any cell grown in culture, whether of plant or animal origin,
vertebrate or invertebrate, and of any tissue or type. In preferred
embodiments, the cells will be animal cells, more preferably
mammalian cells, and most preferably human cells.
Contact between the cells and the nucleic acid-lipid particles,
when carried out in vitro, takes place in a biologically compatible
medium. The concentration of particles varies widely depending on
the particular application, but is generally between about 1
.mu.mol and about 10 mmol. Treatment of the cells with the nucleic
acid-lipid particles is generally carried out at physiological
temperatures (about 37.degree. C.) for periods of time of from
about 1 to 48 hours, preferably of from about 2 to 4 hours.
In one group of preferred embodiments, a nucleic acid-lipid
particle suspension is added to 60-80% confluent plated cells
having a cell density of from about 10.sup.3 to about 10.sup.5
cells/mL, more preferably about 2.times.10.sup.4 cells/mL. The
concentration of the suspension added to the cells is preferably of
from about 0.01 to 0.2 .mu.g/mL, more preferably about 0.1
.mu.g/mL.
The nucleic acid-lipid particles of the present invention can be
adsorbed to almost any cell type with which they are mixed or
contacted. Once adsorbed, the particles can either be endocytosed
by a portion of the cells, exchange lipids with cell membranes, or
fuse with the cells. Transfer or incorporation of the nucleic acid
portion of the particle can take place via any one of these
pathways. In particular, when fusion takes place, the particle
membrane is integrated into the cell membrane and the contents of
the particle combine with the intracellular fluid.
Using an Endosomal Release Parameter (ERP) assay, the delivery
efficiency of the SNALP or other lipid-based carrier system can be
optimized. An ERP assay is described in detail in U.S. Patent
Publication No. 20030077829. More particularly, the purpose of an
ERP assay is to distinguish the effect of various cationic lipids
and helper lipid components of SNALPs based on their relative
effect on binding/uptake or fusion with/destabilization of the
endosomal membrane. This assay allows one to determine
quantitatively how each component of the SNALP or other lipid-based
carrier system effects delivery efficiency, thereby optimizing the
SNALPs or other lipid-based carrier systems. Usually, an ERP assay
measures expression of a reporter protein (e.g., luciferase,
.beta.-galactosidase, green fluorescent protein, etc.), and in some
instances, a SNALP formulation optimized for an expression plasmid
will also be appropriate for encapsulating an interfering RNA. In
other instances, an ERP assay can be adapted to measure
downregulation of transcription or translation of a target sequence
in the presence or absence of an interfering RNA. By comparing the
ERPs for each of the various SNALPs or other lipid-based
formulations, one can readily determine the optimized system, e.g.,
the SNALP or other lipid-based formulation that has the greatest
uptake in the cell.
Suitable labels for carrying out the ERP assay of the present
invention include, but are not limited to, spectral labels, such as
fluorescent dyes (e.g., fluorescein and derivatives, such as
fluorescein isothiocyanate (FITC) and Oregon Green.TM.; rhodamine
and derivatives, such Texas red, tetrarhodimine isothiocynate
(TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA,
CyDyes.TM., and the like; radiolabels, such as .sup.3H, .sup.125I,
35S, .sup.14C, .sup.32P, .sup.33P, etc.; enzymes, such as horse
radish peroxidase, alkaline phosphatase, etc.; spectral
colorimetric labels, such as colloidal gold or colored glass or
plastic beads, such as polystyrene, polypropylene, latex, etc. The
label can be coupled directly or indirectly to a component of the
SNALP or other lipid-based carrier system using methods well known
in the art. As indicated above, a wide variety of labels can be
used, with the choice of label depending on sensitivity required,
ease of conjugation with the SNALP component, stability
requirements, and available instrumentation and disposal
provisions.
B. In vivo Delivery
The nucleic acid-lipid particles of the present invention can be
administered via any route known in the art including, e.g.,
intravenously, intramuscularly, subcutaneously, intradermally,
intraperitoneally, orally, intranasally, or topically either alone
or in mixture with a physiologically-acceptable carrier (such as
physiological saline or phosphate buffer) selected in accordance
with the route of administration and standard pharmaceutical
practice.
When preparing pharmaceutical preparations of the nucleic
acid-lipid particles of the invention, it is preferable to use
quantities of the nucleic acid-lipid particles which have been
purified to reduce or eliminate empty lipid particles or particles
with nucleic acid portion associated with the external surface. The
pharmaceutical carrier is generally added following particle
formation. Thus, after the particle is formed, the particle can be
diluted into pharmaceutically acceptable carriers.
The concentration of particles in the pharmaceutical formulations
can vary widely, i.e., from less than about 0.05%, usually at or at
least about 2.5% to as much as 10 to 30% by weight and will be
selected primarily by fluid volumes, viscosities, etc., in
accordance with the particular mode of administration selected.
1. Injectable Delivery
In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein parenterally,
intravenously, intramuscularly, subcutaneously, intradermally, or
intraperitoneally as described in U.S. Pat. Nos. 5,543,158;
5,641,515 and 5,399,363. Solutions of the nucleic acid-lipid
particles may be prepared in water suitably mixed with a
surfactant. Dispersions may also be prepared in glycerol, liquid
polyethylene glycols, and mixtures thereof and in oils. Typically,
these preparations contain a preservative to prevent the growth of
microorganisms. Generally, when administered intravenously, the
nucleic acid-lipid particles formulations are formulated with a
suitable pharmaceutical carrier. Generally, normal buffered saline
(135-150 mM NaCl) will be employed as the pharmaceutically
acceptable carrier, but other suitable carriers will suffice.
Additional suitable carriers are described in, e.g., REMINGTON'S
PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia,
Pa., 17th ed. (1985). As used herein, "carrier" includes any and
all solvents, dispersion media, vehicles, coatings, diluents,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, buffers, carrier solutions, suspensions, colloids,
and the like. The phrase "pharmaceutically-acceptable" refers to
molecular entities and compositions that do not produce an allergic
or similar untoward reaction when administered to a human. The
preparation of an aqueous composition that contains a protein as an
active ingredient is well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to injection can also be prepared. The
preparation can also be emulsified.
These compositions can be sterilized by conventional liposomal
sterilization techniques, such as filtration. The compositions may
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents and the like. These compositions can be sterilized using the
techniques referred to above or, alternatively, they can be
produced under sterile conditions. The resulting aqueous solutions
may be packaged for use or filtered under aseptic conditions and
lyophilized, the lyophilized preparation being combined with a
sterile aqueous solution prior to administration.
2. Oral Delivery
In certain applications, the nucleic acid-lipid particles disclosed
herein may be delivered via oral administration to the individual.
The active compounds may even be incorporated with excipients and
used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, mouthwash, suspensions, oral sprays, syrups,
wafers, and the like (see, U.S. Pat. Nos. 5,641,515; 5,580,579 and
5,792,451). The tablets, troches, pills, capsules and the like may
also contain the following: binders, gelatin; excipients,
lubricants, or flavoring agents. When the dosage unit form is a
capsule, it may contain, in addition to materials of the above
type, a liquid carrier. Various other materials may be present as
coatings or to otherwise modify the physical form of the dosage
unit. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed.
Typically, these formulations may contain at least about 0.1% of
the active compound or more, although the percentage of the active
ingredient(s) may, of course, be varied and may conveniently be
between about 1 or 2% and about 60% or 70% or more of the weight or
volume of the total formulation. Naturally, the amount of active
compound(s) in each therapeutically useful composition may be
prepared is such a way that a suitable dosage will be obtained in
any given unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and
treatment regimens may be desirable.
3. Nasal Delivery
In certain embodiments, the pharmaceutical compositions may be
delivered by intranasal sprays, inhalation, and/or other aerosol
delivery vehicles. Methods for delivering nucleic acid compositions
directly to the lungs via nasal aerosol sprays has been described
e.g., in U. S. Pat. Nos. 5,756,353 and 5,804,212. Likewise, the
delivery of drugs using intranasal microparticle resins and
lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are
also well-known in the pharmaceutical arts. Likewise, transmucosal
drug delivery in the form of a polytetrafluoroetheylene support
matrix is described in U.S. Pat. No. No. 5,780,045.
4. Topical Delivery
In another example of their use, nucleic acid-lipid particles can
be incorporated into a broad range of topical dosage forms
including, but not limited to, gels, oils, emulsions and the like.
For instance, the suspension containing the nucleic acid-lipid
particles can be formulated and administered as topical creams,
pastes, ointments, gels, lotions and the like.
EXAMPLES
The following examples are provided to illustrate, but not to limit
the claimed invention.
Example 1
Materials and Methods
Background: Specific gene silencing via RNA interference (RNAi) has
become a widely used tool in biological research and is rapidly
being developed for clinical application. RNAi utilises short
double-stranded RNA (siRNA), 18-22 bp in length, that are widely
regarded as being non-inflammatory and unable to activate the
interferon response in mammalian cells due to their small size.
However, few studies in immunological systems have been reported to
support these contentions. To address this directly, we have
investigated the immunostimulatory properties of a panel of siRNA.
More particularly, chemically synthesised siRNA, either liposome
encapsulated, lipid complexed (Oligofectamine) or naked, were
tested for their ability to stimulate a cytokine response from
human blood cell subsets. Activation of the innate immune system by
siRNA in vivo was also assessed in murine studies.
siRNA: All siRNA used in these studies were chemically synthesized
by Dharmacon (Lafayette, Colo.) and received as desalted,
pre-annealed duplexes in either standard or PAGE-purified formats.
siRNA homologous to mRNA encoding .beta.-galactosidase, firefly
luciferase, BP120 and the bacterial Tetracycline Resistance gene
(TetR) were generated together with corresponding non-targeting
sequence control siRNAs. These nucleotide sequences are detailed in
FIG. 16. .beta.-gal control and BP1 control sequences were modified
by selective base substitutions as described and detailed in FIG.
12A.
Mice: 6-8 week old CD I ICR mice were obtained from Harlan
(Indianapolis Ind.) and subject to a three week quarantine and
acclimation period prior to use. siRNA and lipid formulations were
administered as a single intravenous injection in the lateral tail
vein in 0.2 ml PBS. Injections were administered over a period of
3-5 seconds. Blood was collected by cardiac puncture and processed
as plasma for cytokine analysis. Blood cell counts were performed
at The Central Laboratory For Veterinarians (Langley, BC).
Lipid Encapsulation of siRNA: siRNAs were encapsulated into
liposomes by an adaptation of the method developed by Wheeler et
al., Gene Ther. 6, 271-281 (1999), whereby detergents are replaced
by ethanol for the solublization and dialysis of the lipid
components. Liposomes were composed of the following lipids;
synthetic cholesterol (Sigma, St. Louis, Mo.), the phospholipid
DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine; Avanti Polar
Lipids, Alabaster, Ala.), the PEG-lipid PEG-cDMA (3-N-[(-Methoxy
poly(ethylene
glycol)2000)carbamoyl]-1,2-dimyrestyloxy-propylamine), and the
cationic lipid DODMA
(1,2-Di-o-octadecenyl-3-(N,N-dimethyl)aminopropane) or DLinDMA
(1,2-Dilinoleyloxy-3-(N,N-dimethyl)aminopropane) in the molar
ratios 55:20:10:15, or 48:20:2:30 respectively. The lipids
PEG-cDMA, DODMA, and DLinDMA were synthesized in house at Protiva
Biotherapeutics. The resulting stabilized lipid particles were
dialyzed in PBS prior to use. For vehicle controls, empty liposomes
with identical lipid composition were formed in the absence of
siRNA.
Formation of siRNA Complexes: In some experiments, siRNA were
complexed with either oligofectamine or Lipofectamine (Invitrogen;
Carlsbad, Calif.) according to the manufacturers instructions.
siRNA were complexed with either 10 KDa PEI (Polysciences Inc.
Warrington, Pa.) or poly-L-lysine (Sigma; Poole, UK) diluted in
distilled water by dropwise addition of the nucleic acid to the
polycation solution while vortexing. PEI Polyplexes were formed at
an approximate N:P ratio of 10.5:1 and PLL polyplexes at a charge
ratio of 3:1 (+:-). The resulting polyplexes were approximately 140
nm and >500 nm in diameter respectively.
Cell Isolation and Culture: Human PBMC were isolated from whole
blood from healthy donors by a standard Ficoll-Hypaque density
centrifugation technique. Isolation of CD14.sup.+ monocytes and
BDCA4.sup.+ pDC from human PBMC was performed by positive selection
with MACS magnetic beads using MiniMacs columns (Miltenyi; Auburn,
Calif.) according to the manufacturers instructions. Yields of pDC
enriched cells were typically 0.3 to 0.5% of the total PBMC
population. For stimulation assays, 2.times.10.sup.5 freshly
isolated cells were seeded in triplicate in 96 well plates and
cultured in RPMI 1640 medium with 10% FCS, 2 mM glutamine, 100 U/mL
penicillin and 100 ug/mL streptomycin. siRNA were either liposome
encapsulated or complexed with Oligofectamine (Invitrogen,
Carlsbad, Calif.) then added to cells at the indicated final
nucleic acid concentration. Empty liposomes or Oligofectamine alone
were used as lipid vehicle controls. In some experiments, cultures
were supplemented with 20% autologous human plasma or various
concentrations of chloroquine (Sigma, St. Louis, Mo.) at the start
of culture. Supernatants were collected after 16-20 h of culture
and assayed for IFN-.alpha., IL-6 and TNF-.alpha. by sandwich
ELISA.
Cytokine ELISA: All cytokines were quantified using sandwich ELISA
kits. These were mouse and human interferon-.alpha. (PBL
Biomedical, Piscataway, N.J.), Human IL-6 and TNF-.alpha.
(eBioscience, San Diego, Calif.) and mouse IL-6, TNF-.alpha. and
IFN-.alpha. (BD Biosciences, San Diego, Calif.).
In Vitro RNA Interference Assay: Murine Neuro2a-LacZ cell lines
that stably express .mu.-gal were generated by lipid transfection
of neuro2a cells with a pcDNA5/LacZ construct (Invitrogen, Carlsbad
Calif.). Stable transfectants were selected and maintained using
Hygromycin. LacZ-Neuro2a cells were seeded into 24 well plates and
after overnight culture, treated with lipid encapsulated siRNA
targeting .beta.-gal or the non-targeting sequence control duplex.
Cells were then cultured for a further 48 h before being washed and
lysed with 250 mM sodium phosphate containing 0.1% Triton-X100.
.beta.-galactosidase enzyme activity was quantitated in cell
lysates using the CPRG assay (Gene Therapy Systems, San Diego,
Calif.) according to the manufacturer's instructions. Results from
the CPRG assay were confirmed in parallel experiments by Xgal
staining of fixed cell monolayers and microscopic analysis.
Example 2
SNALP Encapsulating siRNA Exhibit Extended Blood Circulation Times
that are Regulated by the PEG-lipid
Male A/J mice bearing subcutaneous Neuro2a tumors on the hind flank
were treated with a single intravenous injection of SNALP (100
.mu.g siRNA) labeled with the non-exchangeable lipid marker
.sup.3H-cholesteryl hexadecyl ether and containing either PEG-c-DSA
or PEG-c-DMA (C18 or C14 alkyl chain length respectively). Whole
blood samples were monitored for the .sup.3H-cholestryl hexadecyl
ether for 24 hours following intravenous injection of the SNALP.
Error bars represent standard errors of the mean (n=5). 50% of
injected dose remains in the blood after 16 h and 3 h for SNALP
containing PEG-c-DSA or PEG-c-DMA respectively. The results are
shown in FIG. 1.
This example demonstrates that blood circulation times are
influenced by the lipid alkyl chain length of the PEG-lipid, i.e.,
PEG-C-DMA formulations preferentially accumulate within the liver,
whereas PEG-C-DSA formulations accumulate at distal tumor sites.
This property could be used to target lipid encapsulated siRNA to
different sites.
Example 3
SNALP can be Programmed to Target Specific Disease Sites Including
the Liver and Distal Tumour
Biodistribution of radio-labeled SNALP was assessed after 24 h in
the tumour bearing mice described in Example 2. PEG-c-DMA SNALP
show preferential accumulation in the liver (35%) compared to
PEG-c-DSA SNALP (13%). In contrast, PEG-c-DSA SNALP demonstrate
enhanced targeting to the tumour site. The results are shown in
FIG. 2.
Example 4
siRNA Duplexes Stimulate Production of Type I Interferons and
Inflammatory Cytokines in Human Cells
To determine if human immune cells are activated by synthetic
siRNA, we cultured human peripheral blood mononuclear cells (PBMC)
in the presence of siRNA either encapsulated in liposomes or
complexed with the transfection reagent Oligofectamine.
Human PBMC isolated by Ficoll centrifugation were incubated
overnight with either 3 .mu.g/ml .beta.-Gal siRNA (Dharmacon)
encapsulated in SNALP, complexed with Oligofectamine (lipoplex) or
naked siRNA. Levels of Interferon-alpha (IFN.alpha.), IL-6 and
TNF-.alpha. in the culture supernatant were assayed by ELISA.
Encapsulated siRNA stimulated predominant IFNa response whereas
complexed siRNA primarily elicited inflammatory cytokines. Naked
siRNA or the lipid components alone were non-stimulatory at this
concentration. (mean+S.D. of triplicate cultures). siRNA duplexes
that were immunostimulatory in the mouse also induced significant
IFN-.alpha. and inflammatory cytokine release from human PBMC when
intracellular delivery was facilitated by either transfection
method. The results are shown in FIG. 3A.
SNALP encapsulating .beta.-gal siRNA (2 mg/kg, .about.40 mg) or
equivalent doses of naked siRNA or lipids alone were intravenously
administered to ICR mice. Plasma cytokines were assayed 6 h after
administration. Significant induction of IFN.alpha., IL-6 and
TNF.alpha. was elicited by siRNA SNALP. (mean+SD, n=4). The results
are shown in FIGS. 3B-3C.
IFN.alpha. responses to different doses of .beta.-Gal siRNA were
assessed in vivo in ICR mice or in vitro using human PBMC. (1
.mu.g/ml siRNA=.about.75 nM). The results are shown in FIGS.
3D-3E.
Treatment with lipids alone or naked siRNA yielded no detectable
cytokine release. Optimal stimulation of IFN-.alpha. was associated
with encapsulated liposomal delivery of siRNA whereas lipid
complexed siRNA induced a predominantly inflammatory cytokine
response.
The stimulation of human PBMC by siRNA was also dependent on
nucleotide sequence. .beta.gal 728 siRNA duplexes were
significantly more potent at inducing IFN-.alpha. (FIG. 9), IL-6
and TNF-.alpha. compared to the BPI duplex. >The relative
potency of various siRNAs at inducing a cytokine response in human
PBMC was similar to that seen in the mouse, suggesting that the
mechanism of siRNA recognition may be broadly conserved. Initial
experiments demonstrated that immune stimulation by siRNA was
enhanced in whole blood cultures. This effect could be
reconstituted in PBMC cultures by the addition of autologous plasma
(FIG. 9). Even under these culture conditions, only high
concentrations of BP1 siRNA was able to induce IFN-.alpha. release.
The mechanism by which autologous plasma enhances the inflammatory
response to siRNA in vitro but may reflect either the provision of
growth factors for cytokine producing cells or the involvement of a
soluble co-factor in the recognition of siRNA by its cognate
receptor.
Example 5
Immunostimulatory Properties of siRNA Are Characteristic of A
Toll-Like Receptor Mediated Immune Response
Human PBMC were stimulated overnight with siRNA SNALP (3 .mu.g/ml)
in the presence of increasing concentrations of chloroquine. Levels
of IFN.alpha. and IL-6 were assessed in culture supernatants by
ELISA. siRNA induced cytokine release was inhibited by >90% at 2
.mu.gM Chloroquine concentration. (mean+S.D. of triplicate
cultures). The results are shown in FIGS. 4A-B. These results
demonstrate that immune stimulation by siRNA SNALP is highly
sensitive to inhibition by chloroquine, thus implying that the
immune stimulation is mediated via a toll-like receptor.
Human monocytes and plasmacytoid dendritic cells (pDC) were
fractionated from PBMC by magnetic bead separation (Miltenyi).
PBMC, monocyte depleted PBMC, monocyte enriched and pDC enriched
fractions were stimulated with increasing doses of siRNA SNALP
overnight. Treatment with lipids alone or naked siRNA yielded no
detectable cytokine release. Induction of IFNa requires the
presence of pDC. Optimal stimulation of IFN-.alpha. was associated
with encapsulated liposomal delivery of siRNA whereas lipid
complexed siRNA induced a predominantly inflammatory cytokine
response. Cell fractionation studies using magnetic bead separation
revealed BDCA4+plasmacytoid dendritic cells (pDC) to be the primary
PBMC cell type responsible for the IFN-.alpha. response to lipid
encapsulated siRNA. By contrast, purified CD14.sup.+ monocytes
produced little IFN-.alpha. when cultured with stimulatory siRNA
whereas monocyte depleted PBMC retained full capacity to respond.
IFN.alpha. levels represent mean of pooled triplicate cultures.
Data is representative of 3 separate experiments. The results are
shown in FIG. 4C. These results demonstrate that plasmacytoid
dendritic cells are the principal PBMC cell type responsible for
the interferon response to siRNA, further supporting the
possibility that the immune stimulation is mediated via a toll-like
receptor.
Example 6
siRNA-Cationic Lipid Complexes Induce an Immune Response
This example describes experiments demonstrating in vitro induction
of immune responses by siRNA-cationic lipid complexes.
Mouse splenocyte cell suspensions were prepared from ICR mouse
spleens and stimulated with either 1 .mu.g/ml or 3 .mu.g/ml siRNA
complexed with Oligofectamine.TM.. IFN-.alpha. levels were measured
in the culture supernatants after overnight culture.
Specifically, the following siRNA molecules (SEQ ID NOS: 102-113)
were used:
TABLE-US-00001 1. .beta.Ga1 siRNA (Immunostimulatory sequence) 5'-U
U G A U G U G U U U A G U C G C U A U U-3' 3'-U U A A C U A C A C A
A A U C A G C G A U-5' 2. .beta.Ga1 Mod-1 (U .fwdarw. C
substitution) 5'-U U G A U G C G U U U A G U C G C U A U U-3' 3'-U
U A A C U A C G C A A A U C A G C G A U-5' 3. .beta.Ga1 Mod-2 (U
.fwdarw. C; U .fwdarw. C substitutions) 5'-U U G A U G C G C U U A
G U C G C U A U U-3' 3'-U U A A C U A C G C G A A U G A G C G A
U-5' 4. BP1 siRNA (less immunostimulatory) 5'-C A G C U U U G G C U
G A G C G U A U U U-3' 3'-U U G U C G A A A C C G A C U C G C A U
A-5' 5. BP1 Mod-1 (G .fwdarw. U substitution) 5'-C A G C U U U G U
C U G A G C G U A U U U-3' 3'-U U G U C G A A A C A G A C U C G C A
U A-5' 6. BP1 Mod-2 (G .fwdarw. U, C .fwdarw. G substitutions) 5'-C
A G C U U U G U G U G A G C G U A U U U-3' 3'-U U G U C G A A A C A
C A C U C G C A U A-5'
The results are shown in FIG. 5. These results provide support that
GU-rich motifs (e.g., 5'-UGU-3' or 5'-UGUGU-3') are responsible for
the immunostimulatory activity of an siRNA duplex.
Example 7
siRNA Encapsulated in Nucleic Acid-Lipid Particles Induces an
Immune Response
This example describes experiments demonstrating in vivo induction
of immune responses by siRNA encapsulated in nucleic acid-lipid
particles comprising PEG-dimyristyloxypropyl conjugates.
The siRNA molecules described in Example 7 above, were synthesized
and encapsulated in nucleic acid-lipid particles using the methods
described herein. The encapsulated siRNA (i.e., BP-1, BP-1 Mod-1,
BP-1 Mod-2, .beta.gal, .beta.gal Mod-1, or .beta.gal Mod-2) was
administered to mice intravenously. Plasma IL-6, TFN-.alpha., and
IFN-.alpha. levels were measured 6 hours after administration of
the siRNA. The results are shown in FIGS. 6-8.
TABLE-US-00002 # SNALP treatment Group Mice Formulation day
terminate ASSAY A 3 PBS day 0 6 hr Plasma for B 4 10% PEG2000 day 0
6 hr cytokine cDMA .beta.Gal analysis control C 4 10% PEG2000 day 0
6 hr cDMA .beta.Gal Mod 1 D 4 10% PEG2000 day 0 6 hr cDMA .beta.Gal
Mod 2 E 4 10% PEG2000 day 0 6 hr cDMA BP1 control F 4 10% PEG2000
day 0 6 hr cDMA BP1 Mod 1 G 4 10% PEG2000 day 0 6 hr cDMA BP1 Mod
2
A single base substitution to disrupt the 5'-UGUGU-3' motif in the
B-Gal siRNA sequence significantly reduces the immunostimulatory
activity of the resulting siRNA duplex (B-Gal Mod1). Conversely,
stepwise introduction of a 5'-UGUGU-3' motif into the BP-1 control
siRNA sequence generates duplexes with increasing immunostimulatory
activity. (Data represent mean+SD, n=4). The results are shown in
FIGS. 6-8. These results demonstrate that siRNA duplexes can be
rendered more or less immunostimulatory by modifying 5'-UGU-3'
motifs. These results also provide further support that the base
sequence motif 5'-UGU-3' or 5'-UGUGU-3' is responsible for the
immunostimulatory activity of an siRNA duplex.
Example 8
Plasma Derived Factors Enhance the Sequence Specific
Immunostimulatory Effects of siRNA in vitro
Human PBMC were cultured overnight with SNALP containing either the
highly stimulatory B-Gal siRNA or the less stimulatory BP-1 control
siRNA set forth in Example 6 above in the presence or absence of
10% autologous plasma. IFN-.alpha. levels are expressed as
mean+/-SD of triplicate cultures. Human plasma enhances the
stimulatory effects of Bgal siRNA and facilitates low level
IFN.alpha. induction by BP-1 control siRNA at high doses.
The stimulation of human PBMC by siRNA was dependent on nucleotide
sequence. .beta.gal siRNA duplexes were significantly more potent
at inducing IFN-.alpha.), IL-6 and TNF-.alpha. compared to the BP1
duplex. Initial experiments demonstrated that siRNA mediated immune
stimulation was enhanced in whole blood cultures. This effect could
be reconstituted in PBMC cultures by the addition of autologous
plasma. Even under these culture conditions, BP1 siRNA was only
able to induce low levels of IFN-.alpha. These results are shown in
FIGS. 9A-9B.
The relative potency of the various siRNAs is similar to that seen
in the mouse, suggesting that the mechanism of siRNA recognition in
humans and mice may be based on broadly similar nucleotide sequence
patterns.
Example 9
siRNA-Cationic Lipid Complexes and siRNA Encapsulated in Nucleic
Acid-Lipid Particles Induce Immune Responses
Additional siRNA sequences were complexed with cationic lipids or
encapsulated in the nuclec acid-lipid particles described herein.
The complexes were contacted with murine splenocytes as in Example
6 and the encapsulated siRNA were administered to mice as in
Example 7. The results from the in vitro and in vivo experiments
are summarized in FIG. 10.
Example 10
Sequence Dependent Induction of Cytokines by Systemically
Administered siRNA.
To examine whether synthetic siRNA can activate an innate immune
response, we tested a panel of siRNA duplexes for their ability to
elicit a cytokine response in mice. To achieve effective systemic
delivery of siRNA to target cells in vivo, we fully encapsulated
synthetic siRNA within liposomes as described in Example 1. The
resulting 100-120 nm diameter lipid particles protect the
encapsulated siRNA from nuclease degradation, exhibit extended
blood circulation times and are effective at mediating RNAi (FIGS.
1 and 13). Intravenous administration of lipid encapsulated siRNA
targeting either .beta.-galactosidase (.beta.-gal 728), firefly
Luciferase (Luc) or the respective non-targeting sequence control
duplexes induced a significant, dose dependent IFN-.alpha. response
in ICR mice (FIGS. 11A and 11B).
This general observation that synthetic siRNA could be potent
stimulators of an innate cytokine response was confirmed in a
second experiment in which mice were treated intravenously with
siRNA duplexes targeting .beta.-gal or TetR encapsulated in an
alternate liposomal formulation (FIG. 11C). Qualitatively similar
responses were also seen in A/J and C57BI/6 strains of mice.
Treatment with siRNA was associated with the concurrent production
of inflammatory cytokines including TNF-.alpha. and IL-6 (FIG.
11C). Maximum cytokine levels were achieved 6-10 h after siRNA
administration and had fully resolved to background levels within
24 h. This cytokine response was dependent on the siRNA and
required its effective intracellular delivery since neither lipid
carriers or the naked siRNA duplexes at equivalent doses induced
detectable cytokine elevations (FIGS. 11A, 11C, and 11D).
Strikingly, treatment with certain siRNAs, for example duplexes
designed to target the breast cancer associated BP1 protein (see,
e.g., Fu et al., Breast Cancer Res. 5, 82-87 (2003) or its
non-targeting sequence control, induced little or no cytokine
response in mice even when administered in encapsulated form (FIGS.
11A and 11D). Since all of these synthetic siRNA duplexes have
similar chemistries, this finding suggests that the
immunostimulatory activity of an siRNA duplex is a function of its
nucleotide sequence.
Example 11
The Immune Stimulatory Activity of Sima is Modulated By GU-rich
Motifs
Although poorly defined, poly U or U and G rich sequences within
ssRNA oligonucleotides have been identified as contributing to
their immunostimulatory effects (see, e.g., Heil et al., Science
303, 1526-1529 (2004) and Diebold et al., Science 303, 1529-1531
(2004)). Our in vivo and in vitro studies demonstrate that immune
stimulatory activity of siRNA is modulated by GU-rich motifs
Analysis of the six siRNA sequences shown in FIG. 16 initially used
in our studies reveals that the highly stimulatory .beta.gal and
non-targeting control duplexes contain a 5'-UGUGU-3' internal
motif. Since this GU rich motif is not present in the poorly
stimulatory BP-1 or BP-1 control duplexes, we hypothesized that
this may contribute to the immunostimulatory activity of the siRNA.
To test this hypothesis, we designed RNA duplexes containing a
single or double base substitution that incrementally disrupt the
5'-UGUGU-3' motif in the .beta.gal control sequence or introduces
the same motif into the BP-1 control sequence (FIG. 12A). Series 1;
.beta.-gal control (highly stimulatory), .beta.-gal Mod1 (single
base substitution) and .beta.-gal Mod2 (double base substitution).
Series 2; BP1 control (low stimulatory), BP1 Mod1 (single base
substitution) and BP1 Mod2 (double base substitution). Base
substitutions are underlined.
In vivo
Lipid-encapsulated sequence modified siRNA duplexes (50 .mu.g) were
intravenously administered to mice. IFN-.alpha. and IL-6 were
assessed in mouse serum 6 h after administration. The single U to C
base substitution in the .beta.-gal siRNA sequence (.beta.-gal
Mod1) almost completely abolished both the IFN-.alpha. and
inflammatory cytokine response when these duplexes were injected
into mice (FIG. 12B). A second U to C base substitution (.beta.gal
Mod2) that further disrupted the original 5'-UGUGU-3' motif
completely abrogated the systemic cytokine response (FIG. 12B).
Conversely, a single G to U base substitution in the BP1 control
sequence, creating a 5'-UGU-3' motif, rendered the modified RNA
duplex immunostimulatory (BP1 Mod 1). This activity was further
enhanced by a second base substitution (BP1 Mod2) that fully
reconstituted the 5'-UGUGU-3' motif (FIG. 12B).
In vitro
Treatment of human PBMC with the sequence modified BP1 and
.beta.-gal RNA duplexes demonstrated that the immunostimulatory
activity of these siRNA on human immune cells was regulated by the
presence of similar motifs in the siRNA sequence (FIGS. 12C and
12D). These findings support the contention that human and mouse
immune cells can recognize broadly similar siRNA sequence motifs
based on GU rich sequences. It also indicates that the
specificities of this siRNA recognition mechanism are strict enough
to allow its disruption by single base pair substitutions within
putative immunostimulatory motifs.
Systemic inflammatory reactions are often accompanied by a
perturbation of hematological parameters. These effects can include
a transient reduction in leukocyte and platelet numbers due to the
margination of these cells from the peripheral blood. Intravenous
treatment of mice with .beta.-gal and other immunostimulatory
siRNAs resulted in a rapid reduction in platelets and white blood
cells (FIG. 12E) that was attributable to the selective loss of
lymphocytes from the peripheral blood. This reaction was transient;
blood cell numbers returned to baseline levels within 72 h of siRNA
administration. The extent of these toxicities correlated with the
degree of cytokine release induced by each siRNA duplex. Treatment
with sequence modified .beta.-gal RNA duplexes that induced minimal
cytokine release had little or no effect on platelet or white blood
cell counts (FIGS. 12D and E). Qualitatively similar results were
obtained with the BP-1 series of modified siRNA (FIGS. 12C and
12F). These findings demonstrate that the use of synthetic siRNA
with non-stimulatory sequences may alleviate potential toxicities
associated with their systemic administration.
FIGS. 12C and 12D illustrate data demonstrating that similar
sequence motifs regulate the immune stimulatory activity of siRNA
on human cells. IFN-.alpha. induction from human PBMC after
overnight culture with (c) encapsulated sequence modified BP 1 or
(d) .beta.-gal siRNA. Values are mean+SD. of triplicate cultures
and representative of 2 separate experiments. FIG. 12D illustrates
data demonstrating that there is a drop in peripheral white blood
cell and platelet counts associated with administration of
immunostimulatory siRNA is ameliorated by RNA sequence
modifications. Mice were treated with 50 .mu.g encapsulated siRNA
and their peripheral WBC and platelet counts recorded at 48 h after
administration. Immunostimulatory .beta.-gal siRNA caused a
substantial drop in both platelet and WBC numbers. These effects
were ameliorated by the selective base substitutions in .beta.-gal
Mod1 and Mod2 sequences.
Example 12
In vitro Properties of Lipid Encapsulated siRNA
Murine Neuro2a cells stably expressing firefly luciferase were
treated with luciferase siRNA or the non-targeting control siRNA
encapsulated in lipid vesicles. The siRNA sequences are provided in
FIG. 16. All siRNA were synthesized with a 3'-UU overhang on each
strand. Immunostimulatory GU-rich motifs (e.g., 5'-UGU-3' and
5'-UGUGU-3' motifs) are underlined. Liposome encapsulation of siRNA
was performed as described in Example 1 above. Luciferase
expression after 48 h culture is expressed as percent of media only
control cultures. Values are mean+SD of triplicate cultures. The
results are shown in FIG. 13 and demonstrate that lipid
encapsulated siRNA is effective at mediating RNAi in vitro.
Example 13
Lipid-Complexed siRNA and Polycation-Complexed siRNA Induce
Inflammatory Cytokine Responses
In vitro experiments demonstrate that both lipid-complexed siRNA
and polycation-complexed siRNA are immunostimulatory.
Lipid-complexed siRNA
Human PBMC, monocytes or monocyte depleted PBMC fractions were
cultured overnight with Oligofectamine complexed .beta.-gal siRNA.
The inflammatory cytokines, IL-6 and TNF-.alpha., were measured in
the culture supernatants. FIG. 14A illustrates data showing 15
TNF-.alpha.: levels. FIG. 14B illustrates data showing IL-6 levels.
Values are mean of triplicate cultures+/-SD. In a separate
experiment, relatively high doses of lipid complexed siRNA were
able to induce IL-6 and TNF-.alpha. production from purified
monocytes. FIGS. 14A and 14B illustrate data demonstrating that
freshly isolated monocytes can be stimulated with high doses of
lipid-complexed siRNA to produce inflammatory cytokines.
Polycation-complexed siRNA
siRNA Complexed with the Polycations Polyethylenimine or
Poly-L-Lysine Also Activate Potent Cytokine Responses From Human
PBMC. .beta.-gal 728 or .beta.-gal control siRNA were mixed with
either 10 KDa polyethylenimine (PEI) or poly-L-lysine (PLL) as
described in Example 1 above to form polyplexes. Human PBMC were
stimulated with polyplexes at 3 .mu.g/mL siRNA or polycation alone
at equivalent concentrations. IFN-.alpha., IL-6 and TNF-.alpha.
were measured in the culture supernatants after 24 h. Values are
mean+SD of triplicate cultures. All data are representative of at
least 3 separate experiments. FIG. 14C illustrates data
demonstrating that freshly isolated monocytes can be stimulated
with high doses of polycation-complexed siRNA to produce
inflammatory cytokines.
Example 14
Sequence Modification of siRNA Ameliorates their Systemic
Toxicities.
Systemic inflammatory reactions are often accompanied by a
perturbation of hematological parameters. These effects can include
a transient reduction in leukocyte and platelet numbers due to the
margination of these cells from the peripheral blood. Intravenous
treatment of mice with .beta.gal and other immunostimulatory siRNAs
resulted in a rapid reduction in platelets and white blood cells
(FIG. 12E) that was attributable to the selective loss of
lymphocytes from the peripheral blood. This reaction was transient;
blood cell numbers returned to baseline levels within 72 h of siRNA
administration. At higher siRNA doses (5-10 mg/kg; single dose)
more apparent toxicities were observed including body weight loss,
hunched posture and piloerection. These toxicities were dependent
on the encapsulated siRNA and their extent correlated with the
degree of cytokine release induced by each siRNA duplex. Treatment
with sequence modified .beta.-gal RNA duplexes that induced minimal
cytokine release had little or no effect on platelet or white blood
cell counts (FIGS. 12D and 12E) and had no apparent effect on the
general condition of the animal. Qualitatively similar results were
obtained with the BP-1 series of modified siRNA (FIG. 12F). These
findings demonstrate that the use of synthetic siRNA with
non-stimulatory sequences can alleviate potential toxicities
associated with their systemic administration.
Example 15
Immunostimulatory Activity of siRNA is not Caused by Contaminants
Such as ssRNA
To confirm that the immunostimulatory properties of the siRNA
duplexes were not caused by contaminants such as ssRNA, siRNA
duplexes were subjected t polyacrylamide gel electrophoresis (PAGE)
purification and PAGE purification followed by RNAse treatment.
siRNA thus purified and treated was cultured with human PBMC in
vitro or administered to mice in vivo to confirm retention of
immunostimulatory properties. (02461 PAGE purified .beta.-gal
control siRNA duplex or its constituent sense and antisense ssRNA
oligonucleotides were treated for 10 min with 0.5 .mu.g/mL RNase A
in high salt buffer (2 mg/mL RNA in 300mM NaCl) to selectively
degrade ssRNA. RNA samples before and after RNase A treatment run
on 20% non-denaturing polyacrylamide gels (500 ng/lane) confirmed
selective degradation of ssRNA.
.beta.-gal control duplex, GU rich sense and complimentary
antisense ssRNA's were digested with RNase A in high salt buffer (2
mg/mL RNA in 300 mM NaCl) for 5 or 15 mins. RNA was then complexed
with 10 kDa PEI. 200 nM RNA was added to human PBMC cultures and
IFN-.alpha. induction was assayed after overnight culture. FIG. 15B
illustrates data demonstrating that GU rich sense ssRNA induced no
detectable IFN-.alpha. following RNase A treatment and that RNase A
treatment had minimal effect on the induction of IFN-.alpha. by
siRNA duplex compared to untreated samples. Values are mean+SD of
triplicate cultures. Data is representative of 3 separate
experiments.
FIG. 15A illustrates data demonstrating that PAGE purification of
the siRNA duplex does not affect its immunostimulatory activity.
Mice were treated intravenously with 50 .mu.g of standard or PAGE
purified Luciferase siRNA encapsulated in liposomes. Serum
IFN-.alpha. and IL-6 were measured after 6 h. PAGE purification of
the siRNA duplex was performed by Dharmacon (Lafayette, Colo.).
Values are mean+SD (n=4 mice).
Example 16
Stimulation of pDC by siRNA Requires Endosomal Acidification
Human and murine pDC have been identified as the primary producers
of IFN-.alpha. in response to CpG DNA (see, e.g., Hornung et al.,
J. Immunol. 168, 4531-4537 (2002); Kadowaki et al., J. Exp. Med.
194, 863-869 (2001); and Asselin-Paturel et al., J. Immunol. 171,
6466-6477 (2003)) and ssRNA (see, e.g., Diebold et al., 2004, supra
and Heil et al., Science 303, 1526-1529 (2004)) due to their
selective expression of TLR9 and TLR7, respectively. Cell
fractionation studies using magnetic bead separation revealed
BDCA4+pDC (see, e.g., Dzionek et al., J. Immunol. 165, 6037-6046
(2000) and Jego et al., Immunity 19, 225-234 (2003)) to be the
primary human PBMC cell type responsible for the IFN-.alpha.
response to lipid encapsulated siRNA (FIG. 9). By contrast,
purified CD 14+ monocytes produced little IFN-.alpha. when cultured
with stimulatory siRNA whereas monocyte depleted PBMC retained full
capacity to respond (FIG. 9). In a separate experiment, relatively
high concentrations of lipid complexed siRNA were able to induce
IL-6 and TNF-.alpha. production from purified monocytes (FIG. 14).
These biases in the immune response to either encapsulated or
complexed siRNA may reflect differences in how charged siRNA
complexes and neutral liposomes are taken up into cells in vitro
and the context in which the siRNA is subsequently presented.
Recognition of nucleic acids by TLRs typically occurs within the
endosomal/lysosomal compartment of cells. This has been
demonstrated for the stimulation of TLR3 (see, e.g., Matsumoto et
al., J. Immunol. 171, 3154-3162 (2003)), TLR7/8 (see, e.g., Diebold
et al., Science 303, 1529-1531 (2004); Lund et al., PNAS USA 101,
5598-5603 (2004); and Heil, F. et al., Eur. J. Immunol. 33,
2987-2997 (2003)) and TLR9 (see, e.g., Ahamad-Nejad et al., Eur. J.
Immunol. 32, 1958-1968 (2002) and Latz et al., Nature Immunol. 5,
190-198 (2004)) by their respective ligands; dsRNA, ssRNA and CpG
DNA. Endosomal TLR signaling can be blocked by the lysosmotropic
agent chloroquine which acts to inhibit endosome acidification
(see, e.g., Yi et al., J. Immunol. 160, 4755-4761 (1998) and Hacker
et al., Embo J. 17, 6230-6240 (1998)). Chloroquine inhibited the
siRNA mediated release of IFN-.alpha. and IL-6 from human PBMC in a
dose dependent manner (FIGS. 4A and 4B). This degree of sensitivity
to chloroquine (IC90.about.2 .mu.M) is in agreement with other
studies using defined nucleic acid based TLR ligands (see, e.g.,
Diebold et al., Science 303, 1529-1531 (2004); Latz et al., 2004,
supra; and Leadbetter et al., Nature 416, 603-607 (2002))
suggesting that synthetic siRNA may also be recognized by an
endosomally located TLR.
Example 17
Lipid Encapsulated siRNA Stimulates Inflammatory Cytokine Release
from RAW 264 Cells in a Sequence Dependent Manner
To confirm that the cytokine response to siRNA in vivo reflects
activation of multiple cell types of which IFN-.alpha. production
by pDC plays a significant but not exclusive role, studies using
the murine monocytic cell line RAW 264 were conducted. RAW 264
cells express a range of immune receptors including functional
TLR7. RAW 264 cells were plated into 96 well plates, allowed to
adhere overnight then treated with lipid encapsulated .beta.-gal
728, .beta.-gal 2891 siRNA or empty liposomes for 48 h. TNF-.alpha.
was assayed in culture supernatants. Values are mean+/-SD of
triplicate cultures. Data are representative of 3 separate
experiments. The results demonstrate that the cytokine response to
siRNA is not limited to pDC cells and are shown in FIG. 18.
Example 18
Rational Design of Non-Stimulatory siRNA with RNAi Activity
To demonstrate the applicability of our findings to the development
of functional, non-immunostimulatory siRNA, we designed a series of
novel siRNA sequences targeting .beta.-gal that avoided GUGU or
poly U motifs (FIG. 17A). The immunostimulatory activity of these
novel siRNA was significantly reduced compared to the .beta.-gal
728 duplex used in initial studies. IFN-.alpha. induction by lipid
encapsulated .beta.-gal 478 siRNA in human PBMC cultures was
reduced approximately 10-fold while .beta.-gal 924 and 2891
duplexes induced no detectable IFN-.alpha.: response even at high
concentrations (FIG. 17B). A similar reduction in the level of
cytokine induction was observed in mice following intravenous
administration. These novel .beta.-gal siRNA possessed functional
RNAi activity. Lipid encapsulated .beta.-gal 478 and 728 siRNA were
equally effective at inhibiting .beta.-gal protein expression in
stably transfected Neuro2a (FIGS. 17C and 17D) and CT-26 cell
lines. In comparison, .beta.-gal 924 and 2891 siRNA were less
potent at mediating RNAi although some degree of target knockdown
was achieved at higher nucleic acid concentrations (FIG. 17C).
The non-targeting sequence control duplex had no effect on
.beta.-gal expression in the neuroblastoma (FIGS. 17C and 17D) or
carcinoma cell lines used in these in vitro studies despite its
potent induction of cytokine responses in immunological systems
(FIGS. 11 and 12).
FIG. 17 illustrates data demonstrating that siRNA can be designed
that are active in mediating RNAi and have minimal capacity to
activate innate immune responses. FIG. 17A sets forth siRNA
sequences designed to target .beta.-gal (codon start sites 478,
924, and 2891) that lack putative immunostimulatory motifs. FIG.
17B illustrates data demonstrating the immunostimulatory activity
of novel .beta.-gal siRNA. Interferon-.gamma. induction from human
PBMC cultured overnight with lipid-encapsulated .beta.-gal 728,
481, 478, 924, or 2891 siRNA duplexes. Values are from pooled
triplicate cultures at each nucleic acid concentration. Data is
representative of 2 separate experiments. FIG. 17C illustrates data
demonstrating inhibition of Regal activity by novel .beta.-gal
targeting siRNA. Neuro2a-LacZ cells that stably express .beta.-gal
protein were cultured for 48 h with lipid encapsulated .beta.-gal
siRNA or non-targeting control siRNA. .beta.-gal enzyme activity
was assayed in cell lysates and expressed as percent of media only
control cultures. nd=no detectable .beta.-gal activity. Values
represent mean+/-SD of triplicate cultures. Data is representative
of 3 separate experiments. FIG. 17D illustrates data demonstrating
inhibition of .beta.-gal activity by GU-rich .beta.-gal targeting
siRNA. Lipid encapsulated .beta.-gal siRNA or non-targeting
.beta.-gal control siRNA (100 nM) were cultured for 48 h with
either Neuro2a LacZ clone 1 or clone 3 cells that express 2534
+/-334 and 1030+/-118 mU .beta.-gal /mg protein respectively.
.beta.-gal enzyme activity was assayed in cell lysates and
expressed as percent of media only control cultures. nd=no
detectable .beta.-gal activity. Values represent mean+/-SD of
triplicate cultures. Data is representative of 3 separate
experiments.
These findings demonstrate that the selection of mRNA target
sequences lacking putative immunostimulatory motifs can generate
siRNA duplexes with potent RNAi activity and minimal immune system
stimulation. We suggest that such screening and analyses of siRNA
become an important selection criteria when developing siRNA for in
vivo and therapeutic use.
We have identified a potent mechanism of immune stimulation
triggered by the intracellular delivery of synthetic siRNA to cells
of the innate immune system. This response to the siRNA molecule
leads to the release of inflammatory cytokines and high level
production of type I interferons. Significantly, highly stimulatory
siRNA were found to activate both freshly isolated human PBMC in
vitro (<0.1 .mu.g/mL; .about.7.5 nM) and the mouse immune system
in vivo (<1 .mu.g; .about.0.05 mg/kg) at concentrations
routinely employed in RNAi studies to achieve effective knockdown
of the target protein. These findings have significant implications
for the development of siRNA for in vivo use due to the potential
for off target gene effects and toxicities associated with
inflammatory responses and the induction of cytokines.
As a hallmark of viral infection, dsRNA can activate several host
defense mechanisms including TLR3 (see, e.g., Alexopoulou et al.,
Nature 413, 732-738 (2001)), PKR (see, e.g., Saunders and Barber
FASEB J. 17, 961-983 (2003)) and other, as yet defined,
TLR-independent mechanisms (see, e.g., Diebold et al., Nature 424,
324-328 (2003); Hoebe et al., Nat. Immunol. 4, 1223-1229 (2003);
and Akira and Takeda, Nature Rev. Immunol. 4, 499-511 (2004)).
Recent evidence suggests that both synthetic and vector-derived
siRNA molecules have the potential to activate PKR (see, e.g.,
Sledz et al., Nature Cell Biol. 5, 834-839 (2003); Bridge et al.,
Nature genetics 34, 263-264 (2003); and Kim et al., Nat.
Biotechnol. 22, 321-325 (2004)) or TLR3-mediated pathways (see,
e.g., (see, e.g., Kariko et al., J. Immunol. 172, 6545-6549 (2004))
in vitro particularly at high nucleic acid concentrations.
Activation of these pathways however is not considered to be
dependent on the specific nucleotide sequence of the RNA. This is
in striking contrast to the immune response elicited by synthetic
siRNA in our studies that was strictly dependent on the nucleotide
sequence of the siRNA duplex and could be elicited by relatively
low doses of nucleic acid.
By selective base substitutions, we have defined putative
immunostimulatory sequence motifs within siRNA duplexes. These are
based on G and U rich regions exemplified by the 5'-UGUGU-3' motif
identified in the .beta.-gal 728, .beta.-gal control and BP1 Mod 2
RNA duplexes. A single base substitution to disrupt this motif
(.beta.-gal Mod 1 RNA) resulted in a duplex with significantly
lower immunostimulatory capacity, thus highlighting the role of
this motif in activating immune cells that take up the siRNA.
Results from modifying the non-immunostimulatory BP-1 control siRNA
by a single base substitution (see FIG. 12) suggest that the
inclusion of a single GU-rich motif (e.g., a 5'-UGU-3' motif)
within the siRNA can be sufficient to render the duplex
immunostimulatory. It is of note that the two Luc siRNA sequences
that induce moderate cytokine production also contain 5'-UGU-3'
motifs (FIG. 16). The effects of relatively minor sequence
modifications on the immunostimulatory activity of siRNA is further
demonstrated by the comparison of .beta.-gal 478 and .beta.-gal 481
(FIG. 17B) whose mRNA target sequences overlap by 16 of 19 bases
(FIG. 17A, FIG. 16). The resulting siRNA sequences differ by only
three terminal base pairs, however .beta.-gal 481 is a
significantly more potent cytokine inducer compared to .beta.-gal
478. We speculate that this difference results from the
introduction of a U-rich 3' terminus in the .beta.-gal 481 duplex
based on previous observations that poly U RNA species can be
immunostimulatory (see, e.g., Diebold et al., Science 303,
1529-1531 (2004)).
Taken together, our findings indicate that the immunostimulatory
sequence motifs in siRNA are likely to occur at relatively high
frequency in conventionally designed synthetic siRNA. This is
supported by data reported here on 16 RNA duplexes and our analysis
of more than 20 additional siRNA against diverse targets in which a
certain degree of immune activation by the siRNA is the norm rather
than the exception, especially at high nucleic acid concentrations.
We have demonstrated in these studies that the design of siRNA
duplexes that are both active in mediating RNAi and have minimal or
no detectable capacity to activate innate immune responses is
feasible based on target sequence selection.
The nature of the immune response induced by synthetic siRNA shares
many of the hallmarks associated with TLR-mediated recognition of
nucleic acids. These include the requirement for endosomal
acidification (see, e.g., Diebold et al., Science 303, 1529-1531
(2004); Lund et al., PNAS USA 101, 5598-5603 (2004); Yi et al., J.
Immunol. 160, 4755-4761 (1998); and Hacker et al., Embo J. 17,
6230-6240 (1998)) and the rapid activation of pDC to produce high
levels of IFN-.alpha.. Human and murine pDC have been identified as
the primary producers of IFN-.alpha. in response to CpG DNA (see,
e.g., Hornung, et al., J. Immunol. 168, 4531-4537 (2002); Kadowaki
et al., J. Exp. Med. 194, 863-869 (2001); and Asselin-Paturel et
al., J. Immunol. 171, 6466-6477 (2003)) and ssRNA (see, e.g., Heil
et al., Science 303, 1526-1529 (2004); Diebold et al., Science 303,
1529-1531 (2004); and Lund et al., PNAS USA 101, 5598-5603 (2004))
due to their selective expression of TLR9 and TLR7. ssRNA has also
been demonstrated to activate human immune cells through TLR8 (see,
e.g., Heil et al., Science 303, 1526-1529 (2004)) although this
receptor is not considered to be expressed constitutively by human
pDC (see, e.g., Hornung, et al., J. Immunol. 168, 4531-4537 (2002)
and Kadowaki et al., J. Exp. Med. 194, 863-869 (2001)). Given the
characteristics of the immune response to siRNA and the broad
similarities in sequence requirements, we hypothesize that
double-stranded RNA molecules such as siRNA, as well as ssRNA
oligonucleotides, can also be a ligand for TLR7 within the
endosomal compartment. This scenario would be analogous to the
recognition of CpG motifs in the context of either single and
double stranded DNA by TLR9 (see, e.g., Krieg, Annu. Rev. Immunol.
20, 709-760 (2002)). Confirmation of the molecular basis for siRNA
recognition by the innate immune system will be of significant
benefit in further understanding how such responses can be
regulated by modifications of the siRNA duplex.
The potential for synthetic siRNA duplexes to be immunostimulatory
must be taken into consideration when utilizing siRNA for in vivo
applications. Our identification of putative immunostimulatory
sequence motifs within siRNA provides a basis for the rational
design of synthetic siRNA that avoid activation of the innate
immune response and therefore minimize the potential for off target
effects and immunotoxicities. Provided such responses can be
regulated, it can also be envisioned that the stimulatory
properties of an siRNA may be exploited therapeutically, for
example in antiviral indications, where siRNA mediated viral
suppression combined with the local induction of interferons may be
considered beneficial.
Thus, these data demonstrate that siRNA molecules can be potent
activators of innate immunity. Although the mechanism
siRNA-mediated immune stimulation has not been completely
elucidated, the experiments described herein implicate Toll-Like
Receptors. These findings have significant implications for the
clinical development of RNAi as a novel therapeutic approach and in
the interpretation of specific gene silencing effects using
siRNA.
It is to be understood that the above description is intended to be
illustrative and not restrictive. Many embodiments will be apparent
to those of skill in the art upon reading the above description.
The scope of the invention should, therefore, be determined not
with reference to the above description, but should instead be
determined with reference to the appended claims, along with the
full scope of equivalents to which such claims are entitled. The
disclosures of all articles and references, including patent
applications, patents, PCT publications, and Accession Nos. are
incorporated herein by reference for all purposes.
SEQUENCE LISTINGS
1
113121RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-Luc (luciferase) target sequence 1aagauuaugu
ccgguuaugu a 21221RNAArtificial SequencesiRNA (small interfering
RNA, short interfering RNA) Anti-Luc (luciferase) sense sequence
2gauuaugucc gguuauguau u 21321RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-Luc (luciferase)
antisense sequence 3nacauaaccg gacauaaucu u 21421RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
non-specific Luc (luciferase) control target sequence 4aaauguauug
gccuguauua g 21521RNAArtificial SequencesiRNA (small interfering
RNA, short interfering RNA) non-specific Luc (luciferase) control
sense sequence 5auguauuggc cuguauuagu u 21621RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
non-specific Luc (luciferase) control antisense sequence
6nuaauacagg ccaauacauu u 21721RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-beta-gal
(beta-galactosidase) target sequence 7aacuacacaa aucagcgauu u
21821RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-beta-gal (beta-galactosidase) sense sequence
8cuacacaaau cagcgauuuu u 21921RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-beta-gal
(beta-galactosidase) antisense sequence 9naaucgcuga uuuguguagu u
211021RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) non-specific beta-gal (beta-galactosidase) control
target sequence 10aauagcgacu aaacacauca a 211121RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
non-specific beta-gal (beta-galactosidase) control sense sequence
11uagcgacuaa acacaucaau u 211221RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) non-specific beta-gal
(beta-galactosidase) control antisense sequence 12nugauguguu
uagucgcuau u 211321RNAArtificial SequencesiRNA (small interfering
RNA, short interfering RNA) Anti-beta-gal (beta-galactosidase) Mod
1 target sequence 13aauagcgacu aaacgcauca a 211421RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-beta-gal (beta-galactosidase) Mod 1 sense sequence
14uagcgacuaa acgcaucaau u 211521RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-beta-gal
(beta-galactosidase) Mod 1 antisense sequence 15uugaugcguu
uagucgcuau u 211621RNAArtificial SequencesiRNA (small interfering
RNA, short interfering RNA) Anti-beta-gal (beta-galactosidase) Mod
2 target sequence 16aauagcgacu aagcgcauca a 211721RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-beta-gal (beta-galactosidase) Mod 2 sense sequence
17uagcgacuaa gcgcaucaau u 211821RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-beta-gal
(beta-galactosidase) Mod 2 antisense sequence 18uugaugcgcu
uagucgcuau u 211921RNAArtificial SequencesiRNA (small interfering
RNA, short interfering RNA) Anti-breast cancer associated BP1-23
target sequence 19aacagcuuug gagccuggua u 212021RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-breast cancer associated BP1-23 sense sequence 20cagcuuugga
gccugguauu u 212121RNAArtificial SequencesiRNA (small interfering
RNA, short interfering RNA) Anti-breast cancer associated BP1-23
antisense sequence 21nuaccaggcu ccaaagcugu u 212221RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-breast cancer associated BP1-23 control target sequence
22aacagcuuug gcugagcgua u 212321RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-breast cancer
associated BP1-23 control sense sequence 23cagcuuuggc ugagcguauu u
212421RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-breast cancer associated BP1-23 control
antisense sequence 24nuacgcucag ccaaagcugu u 212521RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-breast cancer associated BP1-23 Mod 1 target sequence
25aacagcuuug ucugagcgua u 212621RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-breast cancer
associated BP1-23 Mod 1 sense sequence 26cagcuuuguc ugagcguauu u
212721RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-breast cancer associated BP1-23 Mod 1
antisense sequence 27auacgcucag acaaagcugu u 212821RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-breast cancer associated BP1-23 Mod 2 target sequence
28aacagcuuug ugugagcgua u 212921RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-breast cancer
associated BP1-23 Mod 2 sense sequence 29cagcuuugug ugagcguauu u
213021RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-breast cancer associated BP1-23 Mod 2
antisense sequence 30auacgcucac acaaagcugu u 213121RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-TetR 57 (Tetracycline Resistance gene) target sequence
31aaggucggaa ucgaagguuu a 213221RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-TetR 57 (Tetracycline
Resistance gene) sense sequence 32ggucggaauc gaagguuuau u
213321RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-TetR 57 (Tetracycline Resistance gene)
antisense sequence 33naaaccuucg auuccgaccu u 213421RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-TetR 547 (Tetracycline Resistance gene) target sequence
34aagagccagc cuucuuauuc g 213521RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-TetR 547 (Tetracycline
Resistance gene) sense sequence 35gagccagccu ucuuauucgu u
213621RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-TetR 547 (Tetracycline Resistance gene)
antisense sequence 36ngaauaagaa ggcuggcucu u 213721RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-TetR 1 (Tetracycline Resistance gene) target sequence
37aaugauagua ugccgccauu a 213821RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-TetR 1 (Tetracycline
Resistance gene) sense sequence 38ugauaguaug ccgccauuau u
213921RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-TetR 1 (Tetracycline Resistance gene)
antisense sequence 39naauggcggc auacuaucau u 214021RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
control TetR (Tetracycline Resistance gene) target sequence
40aaggucggag cuaaagguuu a 214121RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) control TetR (Tetracycline
Resistance gene) sense sequence 41ggucggagcu aaagguuuau u
214221RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) control TetR (Tetracycline Resistance gene)
antisense sequence 42naaaccuuua gcuccgaccu u 214321RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-TetR 50 (Tetracycline Resistance gene) target sequence
43aauuaaugag gucggaaucg a 214421RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-TetR 50 (Tetracycline
Resistance gene) sense sequence 44uuaaugaggu cggaaucgau u
214521RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-TetR 50 (Tetracycline Resistance gene)
antisense sequence 45ncgauuccga ccucauuaau u 214621RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-TetR 324 (Tetracycline Resistance gene) target sequence
46aaacaguaug aaacucucga a 214721RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-TetR 324 (Tetracycline
Resistance gene) sense sequence 47acaguaugaa acucucgaau u
214821RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-TetR 324 (Tetracycline Resistance gene)
antisense sequence 48nucgagaguu ucauacuguu u 214921RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
Anti-TetR 425 (Tetracycline Resistance gene) target sequence
49aauagguugc guauuggaag a 215021RNAArtificial SequencesiRNA (small
interfering RNA, short interfering RNA) Anti-TetR 425 (Tetracycline
Resistance gene) sense sequence 50uagguugcgu auuggaagau u
215121RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) Anti-TetR 425 (Tetracycline Resistance gene)
antisense sequence 51ncuuccaaua cgcaaccuau u 215221RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA) ALB1#5
target sequence 52aaugaaguug ccagaagaca u 215321RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA) ALB1#5
sense sequence 53ugaaguugcc agaagacauu u 215421RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA) ALB1#5
antisense sequence 54augucuucug gcaacuucau u 215521RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA) ALB1#6
target sequence 55aaugacacca ugccugcuga u 215621RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA) ALB1#6
sense sequence 56ugacaccaug ccugcugauu u 215721RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA) ALB1#6
antisense sequence 57aucagcaggc auggugucau u 215821RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA) ALB1#7
target sequence 58aaagugugca agaacuaugc u 215921RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA) ALB1#7
sense sequence 59agugugcaag aacuaugcuu u 216021RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA) ALB1#7
antisense sequence 60agcauaguuc uugcacacuu u 216121RNAArtificial
SequencesiRNA (small interfering RNA, short interfering RNA)
F4/80#1 target sequence 61aagccaagug cagcugucuu a
216221RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) F4/80#1 sense sequence 62gccaagugca gcugucuuau u
216321RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) F4/80#1 antisense sequence 63uaagacagcu gcacuuggcu
u 216421RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) F4/80#2 target sequence 64aacagcugua ccugucaacc a
216521RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) F4/80#2 sense sequence 65cagcuguacc ugucaaccau u
216621RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) F4/80#2 antisense sequence 66ugguugacag guacagcugu
u 216721RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) F4/80#6 target sequence 67aagaagucug agaggccuau c
216821RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) F4/80#6 sense sequence 68gaagucugag aggccuaucu u
216921RNAArtificial SequencesiRNA (small interfering RNA, short
interfering RNA) F4/80#6 antisense sequence 69gauaggccuc ucagacuucu
u 217019RNAArtificial Sequencebeta-gal (beta-galactosidase) control
siRNA (small interfering RNA, short interfering RNA) 70uugauguguu
uagucgcua 197119RNAArtificial Sequencebeta-gal (beta-galactosidase)
control siRNA (small interfering RNA, short interfering RNA)
71uagcgacuaa acacaucaa 197219RNAArtificial Sequencebeta-gal
(beta-galactosidase) Mod 1 siRNA (small interfering RNA, short
interfering RNA) 72uugaugcguu uagucgcua 197319RNAArtificial
Sequencebeta-gal (beta-galactosidase) Mod 1 siRNA (small
interfering RNA, short interfering RNA) 73uagcgacuaa acgcaucaa
197419RNAArtificial Sequencebeta-gal (beta-galactosidase) Mod 2
siRNA (small interfering RNA, short interfering RNA) 74uugaugcgcu
uagucgcua 197519RNAArtificial Sequencebeta-gal (beta-galactosidase)
Mod 2 siRNA (small interfering RNA, short interfering RNA)
75uagcgacuaa gcgcaucaa 197619RNAArtificial Sequencebreast cancer
associated BP1 control siRNA (small interfering RNA, short
interfering RNA) 76cagcuuuggc ugagcguau 197719RNAArtificial
Sequencebreast cancer associated BP1 control siRNA (small
interfering RNA, short interfering RNA) 77auacgcucag ccaaagcug
197819RNAArtificial Sequencebreast cancer associated BP1 Mod 1
siRNA (small interfering RNA, short interfering RNA) 78cagcuuuguc
ugagcguau 197919RNAArtificial Sequencebreast cancer associated BP1
Mod 1 siRNA (small interfering RNA, short interfering RNA)
79auacgcucag acaaagcug 198019RNAArtificial Sequencebreast cancer
associated BP1 Mod 2 siRNA (small interfering RNA, short
interfering RNA) 80cagcuuugug ugagcguau 198119RNAArtificial
Sequencebreast cancer associated BP1 Mod 2 siRNA (small interfering
RNA, short interfering RNA) 81auacgcucac acaaagcug
198219RNAArtificial Sequenceluciferase siRNA (small interfering
RNA, short interfering RNA) 82gauuaugucc gguuaugua
198319RNAArtificial Sequenceluciferase siRNA (small interfering
RNA, short interfering RNA) 83uacauaaccg gacauaauc
198419RNAArtificial Sequenceluciferase control siRNA (small
interfering RNA, short interfering RNA) 84auguauuggc cuguauuag
198519RNAArtificial Sequenceluciferase control siRNA (small
interfering RNA, short interfering RNA) 85cuaauacagg
ccaauacau 198619RNAArtificial Sequencebeta-gal (beta-galactosidase)
siRNA (small interfering RNA, short interfering RNA) 86cuacacaaau
cagcgauuu 198719RNAArtificial Sequencebeta-gal (beta-galactosidase)
siRNA (small interfering RNA, short interfering RNA) 87aaaucgcuga
uuuguguag 198819RNAArtificial Sequencebeta-gal (beta-galactosidase)
control siRNA (small interfering RNA, short interfering RNA)
88uagcgacuaa acacaucaa 198919RNAArtificial Sequencebeta-gal
(beta-galactosidase) control siRNA (small interfering RNA, short
interfering RNA) 89uugauguguu uagucgcua 199019RNAArtificial
Sequencebreast cancer associated BP1-23 siRNA (small interfering
RNA, short interfering RNA) 90cagcuuugga gccugguau
199119RNAArtificial Sequencebreast cancer associated BP1-23 siRNA
(small interfering RNA, short interfering RNA) 91auaccaggcu
ccaaagcug 199219RNAArtificial Sequencebreast cancer associated
BP1-23 control siRNA (small interfering RNA, short interfering RNA)
92cagcuuuggc ugagcguau 199319RNAArtificial Sequencebreast cancer
associated BP1-23 control siRNA (small interfering RNA, short
interfering RNA) 93auacgcucag ccaaagcug 199419RNAArtificial
Sequencebeta-gal (beta-galactosidase) 728 siRNA (small interfering
RNA, short interfering RNA) 94cuacacaaau cagcgauuu
199519RNAArtificial Sequencebeta-gal (beta-galactosidase) 728 siRNA
(small interfering RNA, short interfering RNA) 95aaaucgcuga
uuuguguag 199619RNAArtificial Sequencebeta-gal (beta-galactosidase)
478 siRNA (small interfering RNA, short interfering RNA)
96gaaggccaga cgcgaauua 199719RNAArtificial Sequencebeta-gal
(beta-galactosidase) 478 siRNA (small interfering RNA, short
interfering RNA) 97uaauucgcgu cuggccuuc 199819RNAArtificial
Sequencebeta-gal (beta-galactosidase) 924 siRNA (small interfering
RNA, short interfering RNA) 98uuaugccgau cgcgucaca
199919RNAArtificial Sequencebeta-gal (beta-galactosidase) 924 siRNA
(small interfering RNA, short interfering RNA) 99ugugacgcga
ucggcauaa 1910019RNAArtificial Sequencebeta-gal
(beta-galactosidase) 2891 siRNA (small interfering RNA, short
interfering RNA) 100ggacgcgcga auugaauua 1910119RNAArtificial
Sequencebeta-gal (beta-galactosidase) 2891 siRNA (small interfering
RNA, short interfering RNA) 101uaauucaauu cgcgcgucc
1910221RNAArtificial Sequencebeta-gal (beta-galactosidase) mismatch
motif siRNA (small interfering RNA, short interfering RNA)
102uugauguguu uagucgcuau u 2110321RNAArtificial Sequencebeta-gal
(beta-galactosidase) mismatch motif siRNA (small interfering RNA,
short interfering RNA) 103uagcgacuaa acacaucaau u
2110421RNAArtificial Sequencebeta-gal (beta-galactosidase) Mod 1
mismatch motif siRNA (small interfering RNA, short interfering RNA)
104uugaugcguu uagucgcuau u 2110521RNAArtificial Sequencebeta-gal
(beta-galactosidase) Mod 1 mismatch motif siRNA (small interfering
RNA, short interfering RNA) 105uagcgacuaa acgcaucaau u
2110621RNAArtificial Sequencebeta-gal (beta-galactosidase) Mod 2
mismatch motif siRNA (small interfering RNA, short interfering RNA)
106uugaugcgcu uagucgcuau u 2110721RNAArtificial Sequencebeta-gal
(beta-galactosidase) Mod 2 mismatch motif siRNA (small interfering
RNA, short interfering RNA) 107uagcgacuaa gcgcaucaau u
2110821RNAArtificial Sequencebreast cancer associated BP1 mismatch
motif siRNA (small interfering RNA, short interfering RNA)
108cagcuuuggc ugagcguauu u 2110921RNAArtificial Sequencebreast
cancer associated BP1 mismatch motif siRNA (small interfering RNA,
short interfering RNA) 109auacgcucag ccaaagcugu u
2111021RNAArtificial Sequencebreast cancer associated BP1 Mod 1
mismatch motif siRNA (small interfering RNA, short interfering RNA)
110cagcuuuguc ugagcguauu u 2111121RNAArtificial Sequencebreast
cancer associated BP1 Mod 1 mismatch motif siRNA (small interfering
RNA, short interfering RNA) 111auacgcucag acaaagcugu u
2111221RNAArtificial Sequencebreast cancer associated BP1 Mod 2
mismatch motif siRNA (small interfering RNA, short interfering RNA)
112cagcuuugug ugagcguauu u 2111321RNAArtificial Sequencebreast
cancer associated BP1 Mod 2 mismatch motif siRNA (small interfering
RNA, short interfering RNA) 113auacgcucac acaaagcugu u 21
* * * * *